Reactivation of a loss-of-function SlGLK2 allele by frame-restoring genome editing in tomato

Reactivation of a loss-of-function SlGLK2 allele by frame-restoring genome editing in tomato | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reactivation of a loss-of-function SlGLK2 allele by frame-restoring genome editing in tomato Kentaro Ezura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8821050/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Plant domestication and improvement processes have inadvertently led to the loss of gene functions that contribute to crop quality. In widely-cultivated tomato ( Solanum lycopersicum ) varieties, a uniform ripening ( u ) mutation, a loss-of-function allele of the Solanum lycopersicum Golden2-like 2 ( SlGLK2 ) gene, has been selected to improve fruit appearance. However, this selection is associated with reduced nutritional quality of the fruit, lowering the levels of sugars, carotenoids, and tocopherols due to impaired plastid development. In this study, the function of SlGLK2 was restored by introducing an additional mutation. A frameshift was introduced via genome editing using the temperature-tolerant Lachnospiraceae bacterium ND 2006 Cas12a ( Lb Cas12a) system. A 13-bp deletion in the linker region of the SlGLK2 protein in the Slglk2-4 line corrected the reading frame and led to enhanced plastid development in the basal part of the fruit. Biochemical and transcriptomic analyses confirmed the functional restoration of SlGLK2 in the Slglk2-4 line. This study provides a model Micro-Tom line for studying plastid biogenesis in tomato fruits and demonstrates the potential of genome editing to revive the latent functions of pseudogenized genes in modern crops, offering a new approach for recovering traits lost during domestication. GOLDEN2-LIKE Fruit development Plastid Tomato (Solanum lycopersicum) Genome editing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Message Restoration of the domestication-related loss-of-function gene, , through the additional mutation by a conventional genome editing technique. Introduction Plant domestication and improvement have focused on selecting traits that are directly noticeable to humans or essential for cultivation under specific conditions. This process has often resulted in the fixation of alleles that enhance agronomic performance at the cost of losing other alleles associated with traits that were not selected for, a phenomenon recognized as domestication-associated genetic trade-offs (Singh and van der Knaap 2022 ; Glaus et al. 2025 ). For instance, traits such as disease resistance (Gao et al. 2019 ), salt stress tolerance (Wang et al. 2020 ), drought tolerance (Li et al. 2024 ), root architecture (Singh et al. 2019 ), and nutritional value of fruit (Powell et al. 2012 ; Nguyen et al. 2014 ; Lupi et al. 2019 ) were overlooked during initial domestication efforts. Although introgression from wild relatives has been used to improve lost traits (Tanksley and McCouch 1997 ), this strategy often introduces undesirable linked loci, making it a complex and occasionally inefficient process. Plastid development in tomato fruits is a notable example of domestication-associated tradeoffs. Plastids contribute not only to photosynthesis but also to the biosynthesis of various nutritional metabolites such as carotenoids, sugars, and vitamins (Li et al. 2022 ; Mesa and Munné-Bosch 2023 ). The GOLDEN2-LIKE (GLK) transcription factors, which belong to the GARP family of MYB transcription factors, play a key role in regulating plastid development (Fitter et al. 2002 ; Waters et al. 2009 ; Zubo et al. 2018 ; Hernández-Verdeja and Lundgren 2024 ). In tomato, SlGLK1 and SlGLK2 transcription factors govern chloroplast development in the leaves and fruits, respectively (Powell et al. 2012 ). The recessive uniform ripening ( u ) allele, a single nucleotide insertion in the coding region of the SlGLK2 gene, leads to a premature stop codon and loss of function, resulting in a reduction in the number of plastids in the fruit (Powell et al. 2012 ; Nguyen et al. 2014 ). Although this mutation has been widely adopted in breeding programs to eliminate the green shoulder phenotype and improve visual uniformity, it compromises the accumulation of key nutritional compounds (Powell et al. 2012 ; Nguyen et al. 2014 ; Lupi et al. 2019 ). Genome editing using site-directed nucleases (SDNs), particularly the Clustered Regularly Interspaced Short Palindromic repeat (CRISPR)/Cas9 system, represents a powerful tool for the precise modification of target genes (Wang and Doudna 2023 ). SDN-based genome editing is categorized into three types: SDN1 to SDN3 (Podevin et al. 2013 ). Most practical applications to date have used the SDN1 category, which involves the induction of single-point mutations or insertions/deletions (InDels) (Menz et al. 2020 ). Various studies have focused on enhancing agronomic traits by inducing loss-of-function mutations in specific target genes (Chen et al. 2019 ; Wang et al. 2019 ). In contrast, reports demonstrating gain-of-function mutations are limited. For instance, CRISPR/Cas9-mediated deletion of the autoinhibitory domain in the glutamate decarboxylase 3 (GAD3) protein enhanced its activity, thereby elevating γ-aminobutyric acid (GABA) level in tomato fruit (Nonaka et al. 2017 ). Similarly, CRISPR/Cas9-mediated editing of the DELLA/PROCERA gene resulted in a gibberellin-responsive, dominant dwarf allele (Zhu et al. 2018 ; Tomlinson et al. 2019 ). A recent study further demonstrated that base editing can repair a deleterious domestication variant, enabling early fruit yields through the modification of shoot and inflorescence architecture (Glaus et al. 2025 ). Genome editing can therefore be applied not only to disrupt functions but also to optimize gene activity. However, the targeted restoration of a loss-of-function allele has rarely been demonstrated. The current study demonstrates that genome editing can be used not only to disrupt but also to restore the gene function lost during tomato domestication. A compensatory frameshift introduced through a temperature-tolerant Lb Cas12a system reconstituted SlGLK2 activity and rescued plastid development in fruits. These findings highlight a conceptual strategy in which genome editing of the SDN1 category can be used to revive gene functions lost during domestication and provide a framework for recovering valuable horticultural traits. Material and methods Plant materials and growth conditions Wild-type (WT) tomatoes ( Solanum lycopersicum ‘Micro-Tom’) were used in this study. They were grown in a constant-temperature cultivation chamber at 24°C with a photoperiod of 16 h light/8 h dark. Vector construction of a genome editing vector All primers used in this study are listed in Table S1 . To construct the pDe-tt Lb Cas12a_NTPII vector, the pDe-CAS9_NTPII vector, which originated from pDe-CAS9 (Addgene plasmid # 61433) (Fauser et al. 2014 ), was linearized by Acs I digestion and purified using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan). A fragment of temperature-tolerant Lb Cas12a (tt Lb Cas12a), harboring the single mutation D156R, was obtained from pDe-EC-tt Lb Cas12a (Schindele and Puchta 2020 ) after Asc I digestion. The pDe-Cas9, pDe-EC-tt Lb Cas12a, and pEn-RZ- Lb -Chimera vectors were kindly provided by Dr. Holger Puchta (Karlsruhe Institute of Technology, Germany) (Schindele and Puchta 2020 ). The pDe-Cas9_NTPII was generated by inserting a synthetic NTPII cassette into the HindIII-digested pDe-Cas9 fragment using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA). The tt Lb Cas12a fragment was purified by gel extraction using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) and inserted into the AscI-digested pDe-CAS9_NTPII vector using Ligation Mix (Takara, Shiga, Japan). Two complementary oligos containing a 24-bp CRISPR RNA (crRNA)-target sequence were annealed and cloned into the Bbs I-digested pEn-RZ- Lb -Chimera vector using Ligation Mix (Takara, Shiga, Japan). The crRNA is under the control of the Arabidopsis U6-26 promoter and flanked by the hammerhead and hepatitis delta virus ribozyme at the 5′ and 3′ sites, respectively. The crRNA cassette was confirmed by restriction enzyme analysis and sequencing. Finally, the crRNA cassette was transferred to the pDe-tt Lb Cas12a_crRNA:SlGLK2_NTPII vector by the LR reaction using the Gateway LR Clonase II (Thermo Fisher Scientific, Waltham, MA, USA). Agrobacterium-mediated transformation and isolation of mutants The recombinant vector pDe-tt Lb Cas12a_crRNA:SlGLK2_NTPII was transformed into Agrobacterium tumefaciens strain GV2260 and transformed into tomato cv. Micro-Tom according to a previously described procedure (Sun et al. 2006 ). In the T 0 generation, T-DNA positive lines were first isolated by polymerase chain reaction (PCR) of the tt Lb Cas12a region in T-DNA using gene-specific primers. To confirm the presence of mutations in SlGLK2 , the sequence was confirmed using PCR amplification of the target region and subsequent direct sequencing of the PCR products. Transgene-negative homozygous alleles were verified in the T 1 generation using PCR-based sequencing. Sequence analysis Conserved domains and disordered regions were searched using InterProScan analysis (Quevillon et al. 2005 ). Nuclear localization signal (NLS) prediction was conducted using LOCALIZER (Sperschneider et al. 2017 ) and NLStradamus (Nguyen Ba et al. 2009 ). AlphaFold3 was used to predict the structure of SlGLK2 protein (Jumper et al. 2021 ). Sub-cloning of SlGLK2 gene and sequencing Total RNA was extracted from immature green fruit of Micro-Tom ( u allele) and CR-Slglk2-4 mutant using an RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands). First-strand cDNA was synthesized using PrimeScript RT reagent (Takara, Shiga, Japan). The full-length coding sequences (CDSs) were amplified from the cDNA mix using KOD ONE (Toyobo, Tokyo, Japan) and gene-specific primers (Table S1 ). To construct pENTR-SlGLK2(u) and pENTR-SlGLK2m, the amplified CDS fragments were cloned into a PCR-amplified pENTR vector using NEBuilder HiFi DNA Assembly Master Mix. The pENTR-SlGLKw construct, in which the functional U allele of the SlGLK2 gene was inserted, was generated by site-directed mutagenesis PCR using the pENTR-SlGLK2(u) plasmid as a template with gene-specific primers. The amplified fragment was then ligated using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA). Subcellular localization and transactivation assays of SlGLK2 To investigate the subcellular localization of wild-type and mutant SlGLK2 proteins, superfolder green fluorescence protein (sfGFP)-tagged expression vectors were constructed using the pGreenII62-SK backbone. First, a synthetic P19 silencing suppressor sequence was cloned into the Xba I and Kpn I sites of pGreenII62-SK (Hellens et al. 2000 ) to generate pGII62SK-P19. The P19 expression cassette was subsequently PCR-amplified and inserted into the BamH I and Hind III sites of the helper plasmid pSoup (Hellens et al. 2000 ) using the NEBuilder HiFi DNA Assembly Master Mix (NEB, MA, USA), resulting in pSoup-P19_K. This construct was introduced into the A. tumefaciens strain GV3101 (MP90) by electroporation and used as the co-infiltration strain. The synthetic sfGFP gene was cloned into the pGreenII62-SK vector. The full-length coding sequences of SlGLK2 (Wid-type-type, u allele mutant, 13-bp deletion allele mutant) were then PCR-amplified and inserted upstream of sfGFP in-frame, producing pGII62SK-SlGLK2 WT :sfGFP, pGII62SK-SlGLK2 u :sfGFP, and pGII62SK-SlGLK2 CR4 :sfGFP, respectively. These constructs were individually transformed into Agrobacterium GV3101, which carried pSoup-P19_K. Agro-infiltration was conducted according to a standard protocol (Hellens et al. 2000 ) as follows. The transformed Agrobacterium strains were grown overnight, and 1 mL of the cultures was inoculated into 20 mL of Luria–Bertani (LB) broth supplemented with 50 mg/L kanamycin, 50 mg/L rifampicin, 10 mg/L gentamicin, and 15 µM acetosyringone. The culture was incubated at 28°C until optical density at 600nm (OD 600 ) reached ~ 0.5. Subsequently, the grown cells were pelleted and resuspended in infiltration buffer (10 mM MgCl 2 , 10 mM MES, and 150 µM acetosyringone, pH = 5.6) to OD 600 = 1.0 and incubated at room temperature (RT) for 3 h in the dark. The suspensions were infiltrated into the abaxial side of the Nicotiana benthamiana leaves. Then, plants were kept in the dark for 24 h at RT and then grown in a growth chamber for 48–72 h. GFP fluorescence was observed after 4 d of incubation using a BX53 microscope (Evident, Tokyo, Japan) using a U-FBNA filter set. Determination of chlorophyll content Immature green fruit at 15 days after anthesis were harvested for total chlorophyll measurements, which were conducted according to a previous protocol (Ezura et al. 2024 ). Transcriptomic analysis The fruit of the immature green stage at 15 d after anthesis was horizontally dissected into three parts: stem, middle, and tip ends. Then, pericarps from the stem end of WT and CR-Slglk2-4 fruit and from the tip end of CR-Slglk2-4 fruit were harvested and immediately frozen in liquid nitrogen. Total RNA was extracted using a Plant RNeasy Extraction Kit (Qiagen, Germany), according to the manufacturer’s instructions. Each triplicate sample contained pericarps from at least three independent fruit. To remove residual genomic DNA, RNA samples were further purified using the RNA Clean & Concentrator Kit-25 (Zymo Research, USA) with DNase I treatment. RNA quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). For RNA-seq analysis, mRNA was captured using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA), and sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Paired-end sequencing (150 base pairs) for three replicates was performed using Illumina NovaSeq X Plus (Illumina, San Diego, CA, USA). Sequencing data were first cleaned using fastp ver. 0. 20.0 (Chen et al. 2018 ). Cleaned reads were pseudo-aligned to transcripts from the Tomato ITAG4.0 genome using Salmon (v1.9.0) (Patro et al. 2017 ). After processing using tximport (Soneson et al. 2015 ), differentially expressed genes (DEGs) between WT and mutants ( CR-Slglk2-4 ) were identified using edgeR (Robinson et al., 2010) under iDEP 2.01 (Ge et al. 2018 ), applying a false discovery rate (FDR) cutoff of < 0.05. Gene Ontology (GO) term enrichment analysis was performed using ShinyGO v0.80 (Ge et al. 2020 ) with a default setting (FDR cutoff < 0.05) for significance, relative to the whole-transcriptome background. For the comparison with the DEGs in the SlGLK2 -overexpression line (Nguyen et al. 2014 ), DEGs in the SlGLK2 -overexpression line were re-analyzed using publicly-available data (SRA079879) through the same workflow. Quantitative real-time PCR Total RNA was extracted from stem end and tip end of immature green fruits at 15 days after pollination of wild-type Micro-Tom ( u allele) and CR-Slglk2-4 line using an RNeasy plant mini kit (Qiagen, Venlo, The Netherlands). Each biological replicate consisted of at least three fruits, and three biological replicates were analyzed. After DNase I treatment, genomic DNA contamination was checked by PCR. Reverse transcription was conducted using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was conducted on a StepOne Real-Time PCR System (Applied Biosystems, CA, USA) using THUNDERBIRD NEXT qPCR Mix (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. SlCAC (Solyc08g006960) was used as an internal control reference (Expósito-Rodríguez et al. 2008). Primer sequences used in this experiment are listed in Table S1 . Relative expression levels were calculated using the 2 –ΔΔCT method (Livak and Schmittgen 2001), with the Micro-Tom ( u allele) stem end sample set to 1. Gene accession numbers Accession numbers for the genes used in this study are: SlGLK2 (Solyc10g008160), SlPORB (Solyc10g006900), SlMPEC (Solyc10g077040), SlGGDR (Solyc03g115980), SlCAB1C (Solyc02g071010), SlLHCB2 (Solyc07g047850), SlLCYE (Solyc12g008980), and CONSTANS-like (Solyc05g009310). Results Genome editing of uniform ripening ( u ) allele by temperature-tolerant Lb Cas12a To determine whether genome editing could restore the function of a gene lost during tomato domestication, the function of the SlGLK2 gene was restored by re-editing the uniform ripening ( u ) allele, which is widely used in cultivated tomato (Powell et al. 2012 ). The u -allele contains a single-base insertion in the coding region of SlGLK2 (Fig. 1 a). The single-base insertion causes a frameshift and premature truncation of SlGLK2, resulting in the loss of its DNA-binding and dimerization domains (Fig. 1 b). AlphaFold2 predictions of full-length SlGLK2 support that the insertion site is located within a disordered region (Fig. S2 ). This prompted us to create a compensatory frameshift near the insertion site and restore the full-length reading frame. The insertion site was located in an A/T-rich sequence, which was difficult to target with the Cas9 protein (even with the NG-Cas9 type Cas9, data not shown). Therefore, we employed the Cas12a system, which is suitable for targeting A/T-rich regions (Li et al. 2020 ). In addition, a mutated version of a Lachnospiraceae bacterium Cas12a, called temperature-tolerant Lb Cas12a (tt Lb Cas12a), which has enhanced activity at low temperatures (Schindele and Puchta 2020 ) was used. Editing with ttLbCas12a generated three T 0 lines carrying mutations near the target site. One of the three lines, CR-Slglk2 #2, displayed green shoulders at the stem end of the mature green (MG) fruit, resembling the U allele (Powell et al. 2012 ; Nguyen et al. 2014 ; Lupi et al. 2019 ). Direct sequencing of the T 0 lines #2 showed biallelic mutations in confirmed biallelic mutations, a 4-bp deletion ( CR-Slglk2-3 allele), and a 13-bp deletion ( CR-Slglk2-4 allele) (Fig. 1 a). The CR-Slglk2-4 allele caused a frameshift that resulted in a normal reading frame, whereas the CR-Slglk2-2 allele caused a frameshift that resulted in another stop codon (Fig. 1 b). Other alleles lacking reading-frame restoration, a 3-bp deletion ( CR-Slglk2-1 allele) or a 5-bp deletion ( CR-Slglk2-2 allele), also produced fruit comparable to those of the original u -allele plant. To confirm that the CR-Slglk2-4 allele was responsible for the green shoulder phenotype, homozygous lines were obtained and analyzed in the T1 generation. The CR-Slglk2-2/CR-Slglk2-2 line produced fruit comparable to the original u allele. In contrast, the CR-Slglk2-4/CR-Slglk2-4 line produced green shoulder fruit similar to that observed in a T 0 plant (Fig. 2 a, b). Chlorophyll content also increased in the CR-Slglk2-4 fruit (Fig. 2 c). Together, these results indicate that the introduction of the 13-bp deletion into the u allele successfully restored SlGLK2 function. Restoration of SlGLK2 function in CR-Slglk2-4 SlGLK2 expression exhibits a latitudinal gradient from the stem to the stylar end of the immature green fruit in tomato (Nguyen et al. 2014 ). This latitudinal gradient was maintained in CR-Slglk2-4 fruits, with higher transcript levels at the stem end than at the stylar end (Fig. 3 a). Furthermore, SlGLK2 transcript levels were consistently higher in CR-Slglk2-4 fruit than in WT at all examined positions, suggesting either an autoregulatory mechanism or a retrograde signal triggered by enhanced plastid development in the fruit. Thus, the restored green shoulder phenotype can be attributed to the recovery of protein function and elevated SlGLK2 expression, which maintains its spatial expression pattern. This result also confirmed that the uniform ripening phenotype of the u allele results from the loss of SlGLK2 protein activity rather than an altered expression pattern. To further validate the restoration of SlGLK2 function, we determined that the mutation did not affect the splicing patterns. Full-length cDNA sequences were amplified from fruit-derived mRNA of wild-type and CR-Slglk2-4 and subsequently confirmed its sequence. According to the sequences and predicted protein sequences (Fig. S2 ), other than the mutation site, the results indicated that the CR-Slglk2-4 mutation had no impact on the overall splicing patterns. GLK transcription factors are GARP transcription activators with a MYB-like DNA-binding domain, a conserved GOLDEN2 C-terminal (GCT) box, and a nuclear localization signal (NLS) sequence in the upstream region of the MYB-like DNA-binding domain (Hall et al. 1998 ; Rossini et al. 2001 ; Fitter et al. 2002 ). The comparison of amino acid sequences and predicted structures showed that the CR-Slglk2-4 mutation was located upstream of the MYB-like DNA-binding domain, in a region poorly conserved among plant orthologs (Fig. S3, S4). NLS predictions using LOCALIZER (Sperschneider et al. 2017 ) and NLStradamus (Nguyen Ba et al. 2009 ) confirmed that the additional mutation did not alter the NLS signal upstream of the MYB-like domain (Fig. S5). Consistently, the sfGFP fusion proteins of SlGLK2 WT and SlGLK2 CR4 were localized to the nucleus, whereas no signal of SlGLK2 u was detected (Fig. S6). Collectively, these results indicate that the CR-Slglk2-4 allele restored nuclear localization of its translated protein. Restoration of SlGLK2-associated gene expression patterns in CR-Slglk2-4 fruits To determine whether SlGLK2 CR4 in the CR-Slglk2-4 line regulates a gene set similar to functional SlGLK2, transcriptome analysis was conducted using stem or tip end tissue from immature green fruits of u allele and CR-Slglk2-4 plants (Table S2 - S4). In comparison with the u allele, 161 genes were upregulated, and 26 genes were downregulated in the stem end of CR-Slglk2-4 fruit (Fig. 3 b). Of the 161 upregulated genes, 62 (%) overlapped with differentially upregulated genes in the SlGLK2 -overexpression line (Fig. 3 c, Table S5), based on reanalysis of published RNA-seq data (Nguyen et al. 2014 ), whereas only three genes were commonly downregulated (Table S6). Notably, 129 of the 161 genes (75.2%) exhibited higher expression in the stem end than tip end of CR-Slglk2-4 fruit, suggesting their gradient expression pattern. Furthermore, 34 out of the 61 genes (53.8%) were identified as potential direct targets of SlGLK2 (Table S6), exhibiting significant binding signals in prior chromatin immunoprecipitation sequence (ChIP-seq) analysis (Tu et al. 2022 ). Gene ontology analysis of the 62 commonly upregulated gene set showed overrepresentation of biological process and cellular componet terms involved in chloroplast- and photosynthesis-associated processes, including “chlorophyll biosynthetic process (GO:0015995)”, “photosynthesis, light harvesting in photosystem I (GO:0009768)”, “photosystem I reaction center (GO:0009538)”, and “chloroplast thylakoid lumen (GO:0009543) (Fig. 3 d; Table S7). To further validate whether these transcriptional changes, we tested the expression of the commonly upregulated genes with SlGLK2-binding signal in the previous ChIP-seq analysis (Tu et al. 2022 ). Seven genes present in both datasets were selected for quantitative RT-PCR analysis, which confirmed their elevated expression in CR-Slglk2-4 fruits (Fig. 4 ). These genes included key components of chlorophyll biosynthesis and light-harvesting complexes, such as protochlorophyllide reductase B ( SlPORB ), Mg-protoporphyrin IX monomethyl ester cyclase ( SlMPEC ), geranylgeranyl diphosphate reductase ( SlGGDR ), chlorophyll a-b binding protein 1C ( SlCAB1C ), light-harvesting complex B2 ( SlLHCB2 ). In addition, lycopene epsilon-cyclase ( SlLCYE ), recently proposed as a potential SlGLK2-regulated gene in the carotenoid pathway (Sun et al. 2025 ), was aloso upregulated in CR-Slglk2-4 . Notably, one transcription factor, CONSTANS-like zinc finger protein (Solyc05g009310), also displayed elevated expression. Discussion In this study, the function of a domestication-associated non-functional SlGLK2 allele was restored using an SDN1-category genome-editing approach (Fig. 5 ). The introduction of a compensatory frameshift into the u allele recovered SlGLK2 protein activity, reinstated the downstream transcriptional program for plastid biogenesis, and restored the green shoulder phenotype of the fruit. These results demonstrate that conventional SDN1-based genome editing can be used not only to disrupt genes to study their function but also to reactivate dormant genes that were lost during domestication and crop improvements. The re-editing of domestication-derived, non-functional alleles as a strategy to reactivate dormant genes Domesticated plants unintentionally lose beneficial genes during domestication. Non-processed pseudogenes arise from duplication in genomic DNA and subsequent disablement, most commonly through disruptive frameshift mutations or premature stop codon formation (Akhunov et al. 2013 ). Although many pseudogenes have lost their function at the translational level due to a frameshift-associated premature stop codon and reduced or undetectable expression levels of their transcripts, a considerable number of pseudogenes are also transcribed (Pink et al. 2011 ; Sisu et al. 2020 ; Feng et al. 2022 ). In tomato, the protein function of the SlGLK2 gene was lost due to truncation of the translated protein by a premature stop codon, whereas the expression level was maintained (Powell et al. 2012 ). Early tomato domestication and improvement have been focused on fruit appearance and yield rather than stress tolerance and fruit nutrition (Powell et al. 2012 ; Bolger et al. 2014 ; Tieman et al. 2017 ; Li et al. 2024 ). For instance, widely-cultivated varieties produce large fruits but are substantially more sensitive to salt stress than their wild ancestors. A major variant, an in-frame, six-base insertion in the coding sequence of the SlHAK20 gene, which encodes a clade IV HAK/KUP/KT transporter, is associated with Na + /K + homeostasis and mediates salt tolerance in tomato (Wang et al. 2020 ). The C-to-T substitution at nucleotide position 265 of SlBBX18 was selected during tomato evolution, increasing drought sensitivity due to premature stop codon formation (Li et al. 2024 ). Furthermore, a deleterious mutation in the SUPPRESSOR OF SP2 ( SSP2 ) gene in cultivated tomato was repaired by base editing, resulting in early fruit yield (Glaus et al. 2025 ). To our knowledge, this is the first report to show that restoring gene function lost during plant domestication or breeding can be achieved using simple deletion-type genome editing of the SDN1-category. In addition to the introduction of gain-of-function mutations targeting transcriptional regulatory regions (Rodríguez-Leal et al. 2017 ; Oliva et al. 2019 ; Wang et al. 2021 ), this approach may provide an alternative method for introducing attractive traits or enhancing target traits in cultivated varieties in a short period. Conventional breeding methods, which involve crossing with wild species to introduce the target locus, are susceptible to phenomena known as linkage drag and hitchhiking, where traits unrelated to the desired phenotypes are inadvertently introduced. This study demonstrates the practical use of genome editing to overcome these limitations. Future research should explore the integration of advanced genome-editing technologies, such as prime editing and CRISPR/Cas-based homologous recombination (HR), which offer precision and range beyond the capabilities of standard SDN1 techniques. Putative downstream factors associated with SlGLK2-dependent transcriptional programs in tomato fruit The GLK family functions as a central regulator of plastid development and coordinates a wide range of plastid-associated processes (Fitter et al. 2002 ; Waters et al. 2009 ; Zubo et al. 2018 ; Hernández-Verdeja and Lundgren 2024 ). In tomato, chloroplasts differentiate into chromoplasts, accumulating health-promoting nutrients such as carotenoids (Sadali et al. 2019 ; Gong et al. 2024 ). Enhancing plastid development in fruits is a promising approach for improving the nutritional quality of fruit-bearing crops (Wang et al. 2008 ; Alves et al. 2020 ). However, the underlying processes of plastid development and differentiation, particularly their regulatory mechanisms, in developing fruits remain unclear. Our comparative transcriptomic analysis identified 62 genes that were commonly upregulated in both CR-Slglk2-4 and SlGLK2ox fruits, representing potential downstream targets of SlGLK2-regulated transcriptional pathway in developing fruit. Notably, 34 of these genes, including well-known photosynthesis-related genes and some regulatory factors, overlapped with previous ChIP-seq data (Tu et al. 2022 ), suggesting potential direct regulation by SlGLK2. Among the commonly upregulated genes, we identified a CONSTANS-like zinc finger protein (Solyc05g009310), which is homologous to Arabidopsis Class III BBX subfamily members (BBX14/15/16/17) (Khanna et al. 2009 ). In Arabidopsis, BBX16, which directly promotes the expression of SUPERROOT 2 (SUR2), which encodes a suppressor of auxin biosynthesis (Zhang et al. 2017 ), is directly regulated by GLK1 during seedling photomorphogenesis (Veciana et al. 2022 ). Additionally, BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence (Buelbuel et al. 2023 ). Supporting this, the senescence-downregulated genes (SDGs), including the light-harvesting complex and PORB, were highly expressed in the CR-Slglk2-4 fruit. In tomato, CONSTANS-like transcription factors, such as SlCOL1 (Solyc02g089540) and SlBBX24 (Solyc06g073180), have been shown to stabilize SlGLK2 protein, thereby enhancing chlorophyll biosynthesis in fruits (Cui et al. 2024 ). Considering that only a few transcriptional regulators were induced in CR-Slglk2-4 fruit, Solyc05g009310 may contribute to plastid biogenesis secondary regulator SlGLK2-associated transcriptional regulation in tomato fruit, although its precise role remains to be determined. Future studies on isolated candidate genes will further elucidate the regulatory mechanisms underlying plastid biogenesis during tomato fruit development. In addition to downstream targets, SlGLK2 transcript level itself was also elevated in the CR-Slglk2-4 fruit. Since SlGLK2 was not identified as a direct transcriptional target of SlGLK2 in a previous ChIP-seq analysis (Tu et al. 2022 ), this increase is unlikely to result from direct autoregulation. Instead, it may reflect indirect positive feedback mediated by plastid-to-nucleus retrograde signaling. Consistent with this hypothesis, previous study in Arabidopsis have shown reduced GLK1 expression in glk1 / glk2 mutant under condition that promotes chloroplast development, suggesting the presence of some positive direct or indirect feedback regulation between GLK function and its own expression (Quevedo et al. 2025 ). Considering this, the enhanced chloroplast biogenesis caused by restored GLK2 function might generate plastid-derived signals that secondarily promote nuclear SlGLK2 expression. Importantly, SlGLK2 expression in CR-Slglk2-4 fruits exhibited a spatial pattern that closely paralleled chlorophyll accumulation along the stem-to-tip axis. This positional concordance supports a model in which local plastid status and retrograde signaling reinforce SlGLK2-regulated transcriptional programs in a position-dependent manner, thereby contributing to the pronounced green shoulder phenotype observed in this line. CR-Slglk2-4 as a model resource for studying plastid biogenesis in tomato fruit The miniature cultivar Micro-Tom has been widely used as a model system for studying fruit physiology and development due to its small plant size, shorter life cycle, and extensive mutant resources (Carvalho et al. 2011 ; Shikata and Ezura 2016 ; Gasparini et al. 2025 ). However, Micro-Tom carries the u allele (Carvalho et al. 2011 ), which limits its utility for study of chloroplast development in fruit. In the CR-Slglk2-4 line, plastid development in fruit is restored within the genetic background of cultivated tomato, resulting in a pronounced green shoulder phenotype. Importantly, this restoration reflects functional recovery of SlGLK2 without introducing transgenes or large-scale genomic modifications. As CR-Slglk2-4 retains the advantageous characteristics of Micro-Tom while exhibiting spatially distinct plastid development within a single fruit, this line provides a useful genetic resource for investigating the regulatory mechanisms underlying plastid biogenesis and its spatial modulation during tomato fruit development. Declarations Acknowledgments We thank Dr. Holger Putcha (Karlsruhe Institute of Technology, Germany) for the pDe-Cas9, pDe-EC-ttLbCas12a, and pEn-RZ-Lb-Chimera vectors. Seed of Micro-Tom NBRP-Japan line (TOMJPF00001) was obtained from the University of Tsukuba, Tsukuba Plant Innovation Research Center, through the National Bio-Resource Project (NBRP) of MEXT/AMED, Japan. We thank Dr. Kiyosada Kawai (Japan International Research Center for Agricultural Sciences, Japan) for kindly sharing Microscopy BX53 (Evident Scientific, Nagano, Japan). We thank all others who kindly shared the experimental tools and resources for this study. This work was financially supported by the Japan Science and Technology Agency (JST) (Grant No. 20J01560 and 24K17885 to K.E.). The author would like to thank Editage (www.editage.com) for English language editing. Data Availability Enquiries about data and materials availability should be directed to the corresponding author. Raw transcriptomic data are deposited at GEO (accession number XXXX). Fundings This work was supported by the Japan Science and Technology Agency (JST) (Grant No. 20J01560 and 24K17885 to K.E.). Competing Interests The author has no relevant financial or non-financial interests to disclose Author contribution K.E. conceptualized and designed the research. K.E. performed all experiments. K.E. wrote and revised the manuscript. K.E. acquired funding. References Akhunov ED, Sehgal S, Liang H, et al (2013) Comparative analysis of syntenic genes in grass genomes reveals accelerated rates of gene structure and coding sequence evolution in polyploid wheat. Plant Physiol 161:252–265. https://doi.org/10.1104/pp.112.205161 Alves FRR, Lira BS, Pikart FC, et al (2020) Beyond the limits of photoperception: constitutively active PHYTOCHROME B2 overexpression as a means of improving fruit nutritional quality in tomato. Plant Biotechnol J 18:2027–2041. https://doi.org/10.1111/pbi.13362 Bolger A, Scossa F, Bolger ME, et al (2014) The genome of the stress-tolerant wild tomato species Solanum pennellii. Nat Genet 46:1034–1038. https://doi.org/10.1038/ng.3046 Buelbuel S, Sakuraba Y, Sedaghatmehr M, et al (2023) Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence. Plant J 116:251–268. https://doi.org/10.1111/tpj.16374 Carvalho RF, Campos ML, Pino LE, et al (2011) Convergence of developmental mutants into a single tomato model system: “Micro-Tom” as an effective toolkit for plant development research. Plant Methods 7:18. https://doi.org/10.1186/1746-4811-7-18 Chen K, Wang Y, Zhang R, et al (2019) Crispr/cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol 70:667–697. https://doi.org/10.1146/annurev-arplant-050718-100049 Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890. https://doi.org/10.1093/bioinformatics/bty560 Cui L, Zheng F, Zhang C, et al (2024) Tomato CONSTANS-LIKE SlCOL1 regulates the content of chlorophyll in fruit by stabilizing the GOLDEN2-LIKE protein. J Integr Agric. https://doi.org/10.1016/j.jia.2024.11.022 Ezura K, Lu Y, Suzuki Y, et al (2024) Class II knotted-like homeodomain protein SlKN5 with BEL1-like homeodomain proteins suppresses fruit greening in tomato fruit. Plant J 118:2037–2054. https://doi.org/10.1111/tpj.16727 Ezura K, Nakamura A, Mitsuda N (2022) Genome-wide characterization of the TALE homeodomain family and the KNOX-BLH interaction network in tomato. Plant Mol Biol 109:799–821. https://doi.org/10.1007/s11103-022-01277-6 Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359. https://doi.org/10.1111/tpj.12554 Feng Y, Wang Z, Chien K-Y, et al (2022) “Pseudo-pseudogenes” in bacterial genomes: Proteogenomics reveals a wide but low protein expression of pseudogenes in Salmonella enterica. Nucleic Acids Res 50:5158–5170. https://doi.org/10.1093/nar/gkac302 Fitter DW, Martin DJ, Copley MJ, et al (2002) GLK gene pairs regulate chloroplast development in diverse plant species. Plant J 31:713–727. https://doi.org/10.1046/j.1365-313x.2002.01390.x Gao L, Gonda I, Sun H, et al (2019) The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor. Nat Genet 51:1044–1051. https://doi.org/10.1038/s41588-019-0410-2 Gasparini K, Figueiredo YG, de Aquino LM, et al (2025) Micro-Tom tomato: from ornamental horticulture to fundamental research. HORTIC ADV 3:8. https://doi.org/10.1007/s44281-025-00062-x Ge SX, Jung D, Yao R (2020) ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics 36:2628–2629. https://doi.org/10.1093/bioinformatics/btz931 Ge SX, Son EW, Yao R (2018) iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics 19:534. https://doi.org/10.1186/s12859-018-2486-6 Glaus AN, Brechet M, Swinnen G, et al (2025) Repairing a deleterious domestication variant in a floral regulator gene of tomato by base editing. Nat Genet 57:231–241. https://doi.org/10.1038/s41588-024-02026-9 Gong J, Li Y, Shen X, et al (2024) Diversity in plastids contributes to variation in fruit color. Sci Hortic 337:113471. https://doi.org/10.1016/j.scienta.2024.113471 Hall LN, Rossini L, Cribb L, Langdale JA (1998) GOLDEN 2: a novel transcriptional regulator of cellular differentiation in the maize leaf. Plant Cell 10:925–936. https://doi.org/10.1105/tpc.10.6.925 Hellens RP, Edwards EA, Leyland NR, et al (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832. https://doi.org/10.1023/a:1006496308160 Hernández-Verdeja T, Lundgren MR (2024) GOLDEN2‐LIKE transcription factors: A golden ticket to improve crops? Plants, People, Planet 6:79–93. https://doi.org/10.1002/ppp3.10412 Jumper J, Evans R, Pritzel A, et al (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589. https://doi.org/10.1038/s41586-021-03819-2 Khanna R, Kronmiller B, Maszle DR, et al (2009) The Arabidopsis B-box zinc finger family. Plant Cell 21:3416–3420. https://doi.org/10.1105/tpc.109.069088 Li J, Ai G, Wang Y, et al (2024) A truncated B-box zinc finger transcription factor confers drought sensitivity in modern cultivated tomatoes. Nat Commun 15:8013. https://doi.org/10.1038/s41467-024-51699-7 Li S, Zhang Y, Xia L, Qi Y (2020) CRISPR-Cas12a enables efficient biallelic gene targeting in rice. Plant Biotechnol J 18:1351–1353. https://doi.org/10.1111/pbi.13295 Li Y, Jian Y, Mao Y, et al (2022) “Omics” insights into plastid behavior toward improved carotenoid accumulation. Front Plant Sci 13:1001756. https://doi.org/10.3389/fpls.2022.1001756 Lupi ACD, Lira BS, Gramegna G, et al (2019) Solanum lycopersicum GOLDEN 2-LIKE 2 transcription factor affects fruit quality in a light- and auxin-dependent manner. PLoS ONE 14:e0212224. https://doi.org/10.1371/journal.pone.0212224 Menz J, Modrzejewski D, Hartung F, et al (2020) Genome edited crops touch the market: A view on the global development and regulatory environment. Front Plant Sci 11:586027. https://doi.org/10.3389/fpls.2020.586027 Mesa T, Munné-Bosch S (2023) α-Tocopherol in chloroplasts: Nothing more than an antioxidant? Curr Opin Plant Biol 74:102400. https://doi.org/10.1016/j.pbi.2023.102400 Nguyen Ba AN, Pogoutse A, Provart N, Moses AM (2009) NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinformatics 10:202. https://doi.org/10.1186/1471-2105-10-202 Nguyen CV, Vrebalov JT, Gapper NE, et al (2014) Tomato GOLDEN2-LIKE transcription factors reveal molecular gradients that function during fruit development and ripening. Plant Cell 26:585–601. https://doi.org/10.1105/tpc.113.118794 Nonaka S, Arai C, Takayama M, et al (2017) Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Sci Rep 7:7057. https://doi.org/10.1038/s41598-017-06400-y Oliva R, Ji C, Atienza-Grande G, et al (2019) Broad-spectrum resistance to bacterial blight in rice using genome editing. Nat Biotechnol 37:1344–1350. https://doi.org/10.1038/s41587-019-0267-z Patro R, Duggal G, Love MI, et al (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417–419. https://doi.org/10.1038/nmeth.4197 Pink RC, Wicks K, Caley DP, et al (2011) Pseudogenes: pseudo-functional or key regulators in health and disease? RNA 17:792–798. https://doi.org/10.1261/rna.2658311 Podevin N, Davies HV, Hartung F, et al (2013) Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding. Trends Biotechnol 31:375–383. https://doi.org/10.1016/j.tibtech.2013.03.004 Powell ALT, Nguyen CV, Hill T, et al (2012) Uniform ripening encodes a Golden 2-like transcription factor regulating tomato fruit chloroplast development. Science 336:1711–1715. https://doi.org/10.1126/science.1222218 Quevedo M, Kubalová I, Brun A, et al (2025) Retrograde signals control dynamic changes to the chromatin state at photosynthesis-associated loci. Nat Commun 16:6527. https://doi.org/10.1038/s41467-025-61831-w Quevillon E, Silventoinen V, Pillai S, et al (2005) InterProScan: protein domains identifier. Nucleic Acids Res 33:W116-20. https://doi.org/10.1093/nar/gki442 Rodríguez-Leal D, Lemmon ZH, Man J, et al (2017) Engineering quantitative trait variation for crop improvement by genome editing. Cell 171:470–480.e8. https://doi.org/10.1016/j.cell.2017.08.030 Rossini L, Cribb L, Martin DJ, Langdale JA (2001) The maize golden2 gene defines a novel class of transcriptional regulators in plants. Plant Cell 13:1231–1244. https://doi.org/10.1105/tpc.13.5.1231 Sadali NM, Sowden RG, Ling Q, Jarvis RP (2019) Differentiation of chromoplasts and other plastids in plants. Plant Cell Rep 38:803–818. https://doi.org/10.1007/s00299-019-02420-2 Schindele P, Puchta H (2020) Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing. Plant Biotechnol J 18:1118–1120. https://doi.org/10.1111/pbi.13275 Shikata M, Ezura H (2016) Micro-Tom Tomato as an Alternative Plant Model System: Mutant Collection and Efficient Transformation. Methods Mol Biol 1363:47–55. https://doi.org/10.1007/978-1-4939-3115-6_5 Singh J, Gezan SA, Vallejos CE (2019) Developmental Pleiotropy Shaped the Roots of the Domesticated Common Bean (Phaseolus vulgaris). Plant Physiol 180:1467–1479. https://doi.org/10.1104/pp.18.01509 Singh J, van der Knaap E (2022) Unintended consequences of plant domestication. Plant Cell Physiol 63:1573–1583. https://doi.org/10.1093/pcp/pcac083 Sisu C, Muir P, Frankish A, et al (2020) Transcriptional activity and strain-specific history of mouse pseudogenes. Nat Commun 11:3695. https://doi.org/10.1038/s41467-020-17157-w Soneson C, Love MI, Robinson MD (2015) Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. [version 2; peer review: 2 approved]. F1000Res 4:1521. https://doi.org/10.12688/f1000research.7563.2 Sperschneider J, Catanzariti A-M, DeBoer K, et al (2017) LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell. Sci Rep 7:44598. https://doi.org/10.1038/srep44598 Sun H-J, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431. https://doi.org/10.1093/pcp/pci251 Sun T, Hazra A, Lui A, et al (2025) GLKs directly regulate carotenoid biosynthesis via interacting with GBFs in plants. New Phytol 246:645–665. https://doi.org/10.1111/nph.20457 Tanksley SD, McCouch SR (1997) Seed banks and molecular maps: unlocking genetic potential from the wild. Science 277:1063–1066. https://doi.org/10.1126/science.277.5329.1063 Tieman D, Zhu G, Resende MFR, et al (2017) A chemical genetic roadmap to improved tomato flavor. Science 355:391–394. https://doi.org/10.1126/science.aal1556 Tomlinson L, Yang Y, Emenecker R, et al (2019) Using CRISPR/Cas9 genome editing in tomato to create a gibberellin-responsive dominant dwarf DELLA allele. Plant Biotechnol J 17:132–140. https://doi.org/10.1111/pbi.12952 Tu X, Ren S, Shen W, et al (2022) Limited conservation in cross-species comparison of GLK transcription factor binding suggested wide-spread cistrome divergence. Nat Commun 13:7632. https://doi.org/10.1038/s41467-022-35438-4 Veciana N, Martín G, Leivar P, Monte E (2022) BBX16 mediates the repression of seedling photomorphogenesis downstream of the GUN1/GLK1 module during retrograde signalling. New Phytol 234:93–106. https://doi.org/10.1111/nph.17975 Wang JY, Doudna JA (2023) CRISPR technology: A decade of genome editing is only the beginning. Science 379:eadd8643. https://doi.org/10.1126/science.add8643 Wang S, Liu J, Feng Y, et al (2008) Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4. Plant J 55:89–103. https://doi.org/10.1111/j.1365-313X.2008.03489.x Wang T, Zhang H, Zhu H (2019) CRISPR technology is revolutionizing the improvement of tomato and other fruit crops. Hortic Res 6:77. https://doi.org/10.1038/s41438-019-0159-x Wang X, Aguirre L, Rodríguez-Leal D, et al (2021) Dissecting cis-regulatory control of quantitative trait variation in a plant stem cell circuit. Nat Plants 7:419–427. https://doi.org/10.1038/s41477-021-00898-x Wang Z, Hong Y, Zhu G, et al (2020) Loss of salt tolerance during tomato domestication conferred by variation in a Na+ /K+ transporter. EMBO J 39:e103256. https://doi.org/10.15252/embj.2019103256 Waters MT, Wang P, Korkaric M, et al (2009) GLK transcription factors coordinate expression of the photosynthetic apparatus in Arabidopsis. Plant Cell 21:1109–1128. https://doi.org/10.1105/tpc.108.065250 Zhang X, Huai J, Shang F, et al (2017) A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis. Plant Physiol 174:2487–2500. https://doi.org/10.1104/pp.17.00418 Zhu Z, Kang X, Lor VS, et al (2018) Characterization of a semidominant dwarfing PROCERA allele identified in a screen for CRISPR/Cas9-induced suppressors of loss-of-function alleles. Plant Biotechnol J 17:319–321. https://doi.org/10.1111/pbi.13027 Zubo YO, Blakley IC, Franco-Zorrilla JM, et al (2018) Coordination of Chloroplast Development through the Action of the GNC and GLK Transcription Factor Families. Plant Physiol 178:130–147. https://doi.org/10.1104/pp.18.00414 Additional Declarations No competing interests reported. Supplementary Files Ezura2026PCRGLK2Supportinginformation1figuresv2.docx Ezura2026PCRGLK2Supportinginformation2tablesv2.xlsx Supplementaryinformation.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 27 Apr, 2026 Reviews received at journal 27 Apr, 2026 Reviews received at journal 26 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 12 Apr, 2026 Reviewers agreed at journal 12 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 09 Feb, 2026 Submission checks completed at journal 09 Feb, 2026 First submitted to journal 08 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Dash indicates deletion. Green bold underlined represents potential stop codons and in-frame stop codons. Purple letter indicates one base pair insertion in the \u003cem\u003euniform ripening\u003c/em\u003e (\u003cem\u003eu\u003c/em\u003e) allele.\u003cstrong\u003e (b) \u003c/strong\u003ePredicted protein domains of wild type (WT) and mutant alleles with disorder region (light blue), nuclear localization signal (pink), a DNA-binding MYB domain (IRR017930) (yellow), and dimerization domain GCT-box (green). aa, amino acids.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/cf539ac2d3e17252d54d38a1.jpg"},{"id":105904232,"identity":"85e0ee7b-9669-4121-b434-ee654798ce46","added_by":"auto","created_at":"2026-04-01 10:06:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGreen shoulder phenotype was restored in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCR-Slglk2-4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mature green (MG) fruits of Micro-Tom (\u003cem\u003eu\u003c/em\u003eallele) and four genome-edited mutant lines at 25 days after anthesis. \u003cstrong\u003e(b)\u003c/strong\u003eThe stem end of MG fruits of Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele) and \u003cem\u003eCR-Slglk2_4\u003c/em\u003eline (\u003cem\u003eCR4\u003c/em\u003e, -13bp) at 25 days after anthesis.\u003cstrong\u003e (c) \u003c/strong\u003eLongitudinal section of MG fruits of Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele) and \u003cem\u003eCR-Slglk2_4\u003c/em\u003e line at 25 days after anthesis.\u003cstrong\u003e (d) \u003c/strong\u003eChlorophyll contents in immature green (IMG) fruit at 15 days after anthesis. Data are means ± SE from three biological replicates. Asterisks indicate significant differences between the genotypes within the same position (an unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test; ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; ns, not significant). Different lowercase letters denote significant differences among positions within Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele), while different uppercase letters denote significant differences among positions within CR4 (one-way ANOVA followed by Tukey’s HSD, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/4fc76842da9d2f126e30bb96.jpg"},{"id":105793668,"identity":"bb497e68-0e9a-43c5-b337-80975cec3511","added_by":"auto","created_at":"2026-03-31 08:20:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":298437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSlGLK2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-associated downstream pathways were enhanced in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCR-Slglk2-4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression of \u003cem\u003eSlGLK2\u003c/em\u003egenes in Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele) and \u003cem\u003eCR-Slglk2-4\u003c/em\u003e line. Data are means ± SE from three biological replicates. Asterisks indicate significant differences between the genotypes within the same position (an unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test; ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not significant). Different lowercase letters denote significant differences among positions within Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele), while different uppercase letters denote significant differences among positions within CR4 (one-way ANOVA followed by Tukey’s HSD, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(b) \u003c/strong\u003eHeat map of differentially expressed genes in stem end of \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit. \u003cstrong\u003e\u0026nbsp;(c) \u003c/strong\u003e61 genes were commonly upregulated in both \u003cem\u003eCR-Slglk2-4\u003c/em\u003e and \u003cem\u003eSlGLK2\u003c/em\u003eox lines compared to Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele). DEGs, differentially expressed genes. \u003cstrong\u003e(d) \u003c/strong\u003eGO overrepresentation analysis of common upregulated genes in \u003cem\u003eCR-Slglk2-4\u003c/em\u003e and \u003cem\u003eSlGLK2\u003c/em\u003eox lines. Photosynthesis- and chlorophyll-related terms were enriched among the common upregulated DEGs. Enriched Gene Ontology (GO) terms in the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) categories are shown. The x-axis indicates −log10(adjusted \u003cem\u003ep\u003c/em\u003e-value), and point size represents fold enrichment of each GO term. Adjusted \u003cem\u003ep\u003c/em\u003e-values were calculated using Bonferroni correction for multiple testing, and terms with an adjusted\u003cem\u003e p\u003c/em\u003e-value \u0026lt; 0.05 were considered significant.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/9e2c593ab6cc4042b4f864e2.jpg"},{"id":105904854,"identity":"73cae8c9-e9ca-4676-9f95-d2437776bef6","added_by":"auto","created_at":"2026-04-01 10:10:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe GLK2-targeted genes were reactivated in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCR-Slglk2-4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e fruit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValidation of expression of common DEGs by quantitative RT-PCR (qRT-PCR). qRT-PCR analysis validates the expression patterns of putative SlGLK2-targeted genes involved in chlorophyll biosynthesis and plastid biogenesis \u003cstrong\u003e(a)\u003c/strong\u003e and a transcription factor \u003cstrong\u003e(b)\u003c/strong\u003e. Data are means ± SE from three biological replicates. Asterisks indicate significant differences between the genotypes within the same position (an unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test; ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not significant). \u003cem\u003eSlPORB\u003c/em\u003e, protochlorophyllide reductase; \u003cem\u003eSlMPEC\u003c/em\u003e, \u003cem\u003eMg-protoporphyrin IX monomethyl ester cyclase\u003c/em\u003e; \u003cem\u003eSlGGDR\u003c/em\u003e, geranylgeranyl diphosphate reductase; \u003cem\u003eSlCAB1C\u003c/em\u003e, chlorophyll a-b binding protein 1C; \u003cem\u003eSlLHCB2\u003c/em\u003e, light-harvesting complex B2; \u003cem\u003eSlLCYE\u003c/em\u003e, lycopene ε-cyclase; \u003cem\u003eSlCO-like\u003c/em\u003e, CONSTANS-like.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/72ef35ea71569a9e98ecde4c.jpg"},{"id":105905159,"identity":"5300efb5-f3cf-4745-ba95-60ad41eddbb2","added_by":"auto","created_at":"2026-04-01 10:11:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic summary of this study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe processes of plant domestication and breeding have inadvertently resulted in trade-offs that affect crop quality. In cultivated tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) varieties, the \u003cem\u003euniform ripening\u003c/em\u003e (\u003cem\u003eu\u003c/em\u003e) mutation, a loss-of-function allele of tomato\u003cem\u003e GOLDEN2-LIKE\u003c/em\u003e (\u003cem\u003eSlGLK2\u003c/em\u003e) characterized by a single base insertion in the coding sequence, has been extensively selected to enhance fruit appearance. However, this mutation reduced the accumulation of sugars, carotenoids, and tocopherol due to impaired plastid development. This study employed a simple CRISPR/Cas system to introduce an additional mutation into the non-functional allele, thereby restoring the function of the lost genes by correcting its reading frame. The 13-bp deletion in the \u003cem\u003eu\u003c/em\u003e allele reinstated \u003cem\u003eSlGLK2\u003c/em\u003e function without altering its spatial expression in fruit and activated the downstream pathway, resulting in a green shoulder phenotype similar to the wild-type \u003cem\u003eU\u003c/em\u003eallele.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/152c38b69af6e90628142d78.jpg"},{"id":105906591,"identity":"fdc1b203-ef94-4a08-8482-3d97d4044fd2","added_by":"auto","created_at":"2026-04-01 10:23:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1591305,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/c2f01c40-d1d0-4bd7-9125-17bcc94eae61.pdf"},{"id":105793662,"identity":"d0c59679-6a68-4e86-9834-8f43578ccc5c","added_by":"auto","created_at":"2026-03-31 08:20:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":940326,"visible":true,"origin":"","legend":"","description":"","filename":"Ezura2026PCRGLK2Supportinginformation1figuresv2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/4f84ef0eb84bedf5a73e2def.docx"},{"id":105904127,"identity":"f234b95f-7afe-4194-8be6-c3d5aec0d25e","added_by":"auto","created_at":"2026-04-01 10:04:41","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4773446,"visible":true,"origin":"","legend":"","description":"","filename":"Ezura2026PCRGLK2Supportinginformation2tablesv2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/56fc22e051156664176f73c7.xlsx"},{"id":105793666,"identity":"9c34e7ca-2087-4f30-ab73-0b285c5e9d66","added_by":"auto","created_at":"2026-03-31 08:20:47","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16196,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8821050/v1/d44179d21e9c9b913cc83f66.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reactivation of a loss-of-function SlGLK2 allele by frame-restoring genome editing in tomato","fulltext":[{"header":"Key Message","content":"\u003cp\u003eRestoration of the domestication-related loss-of-function gene, , through the additional mutation by a conventional genome editing technique.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePlant domestication and improvement have focused on selecting traits that are directly noticeable to humans or essential for cultivation under specific conditions. This process has often resulted in the fixation of alleles that enhance agronomic performance at the cost of losing other alleles associated with traits that were not selected for, a phenomenon recognized as domestication-associated genetic trade-offs (Singh and van der Knaap \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Glaus et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For instance, traits such as disease resistance (Gao et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), salt stress tolerance (Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), drought tolerance (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), root architecture (Singh et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and nutritional value of fruit (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nguyen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lupi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) were overlooked during initial domestication efforts. Although introgression from wild relatives has been used to improve lost traits (Tanksley and McCouch \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), this strategy often introduces undesirable linked loci, making it a complex and occasionally inefficient process.\u003c/p\u003e \u003cp\u003ePlastid development in tomato fruits is a notable example of domestication-associated tradeoffs. Plastids contribute not only to photosynthesis but also to the biosynthesis of various nutritional metabolites such as carotenoids, sugars, and vitamins (Li et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mesa and Munn\u0026eacute;-Bosch \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The GOLDEN2-LIKE (GLK) transcription factors, which belong to the GARP family of MYB transcription factors, play a key role in regulating plastid development (Fitter et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Waters et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zubo et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hern\u0026aacute;ndez-Verdeja and Lundgren \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In tomato, \u003cem\u003eSlGLK1\u003c/em\u003e and \u003cem\u003eSlGLK2\u003c/em\u003e transcription factors govern chloroplast development in the leaves and fruits, respectively (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The recessive \u003cem\u003euniform ripening\u003c/em\u003e (\u003cem\u003eu\u003c/em\u003e) allele, a single nucleotide insertion in the coding region of the \u003cem\u003eSlGLK2\u003c/em\u003e gene, leads to a premature stop codon and loss of function, resulting in a reduction in the number of plastids in the fruit (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nguyen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although this mutation has been widely adopted in breeding programs to eliminate the green shoulder phenotype and improve visual uniformity, it compromises the accumulation of key nutritional compounds (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nguyen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lupi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenome editing using site-directed nucleases (SDNs), particularly the Clustered Regularly Interspaced Short Palindromic repeat (CRISPR)/Cas9 system, represents a powerful tool for the precise modification of target genes (Wang and Doudna \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). SDN-based genome editing is categorized into three types: SDN1 to SDN3 (Podevin et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Most practical applications to date have used the SDN1 category, which involves the induction of single-point mutations or insertions/deletions (InDels) (Menz et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Various studies have focused on enhancing agronomic traits by inducing loss-of-function mutations in specific target genes (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, reports demonstrating gain-of-function mutations are limited. For instance, CRISPR/Cas9-mediated deletion of the autoinhibitory domain in the glutamate decarboxylase 3 (GAD3) protein enhanced its activity, thereby elevating γ-aminobutyric acid (GABA) level in tomato fruit (Nonaka et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similarly, CRISPR/Cas9-mediated editing of the \u003cem\u003eDELLA/PROCERA\u003c/em\u003e gene resulted in a gibberellin-responsive, dominant dwarf allele (Zhu et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tomlinson et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A recent study further demonstrated that base editing can repair a deleterious domestication variant, enabling early fruit yields through the modification of shoot and inflorescence architecture (Glaus et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Genome editing can therefore be applied not only to disrupt functions but also to optimize gene activity. However, the targeted restoration of a loss-of-function allele has rarely been demonstrated.\u003c/p\u003e \u003cp\u003eThe current study demonstrates that genome editing can be used not only to disrupt but also to restore the gene function lost during tomato domestication. A compensatory frameshift introduced through a temperature-tolerant \u003cem\u003eLb\u003c/em\u003eCas12a system reconstituted SlGLK2 activity and rescued plastid development in fruits. These findings highlight a conceptual strategy in which genome editing of the SDN1 category can be used to revive gene functions lost during domestication and provide a framework for recovering valuable horticultural traits.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eWild-type (WT) tomatoes (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e \u0026lsquo;Micro-Tom\u0026rsquo;) were used in this study. They were grown in a constant-temperature cultivation chamber at 24\u0026deg;C with a photoperiod of 16 h light/8 h dark.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVector construction of a genome editing vector\u003c/h3\u003e\n\u003cp\u003eAll primers used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. To construct the pDe-tt\u003cem\u003eLb\u003c/em\u003eCas12a_NTPII vector, the pDe-CAS9_NTPII vector, which originated from pDe-CAS9 (Addgene plasmid # 61433) (Fauser et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), was linearized by \u003cem\u003eAcs\u003c/em\u003eI digestion and purified using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan). A fragment of temperature-tolerant \u003cem\u003eLb\u003c/em\u003eCas12a (tt\u003cem\u003eLb\u003c/em\u003eCas12a), harboring the single mutation D156R, was obtained from pDe-EC-tt\u003cem\u003eLb\u003c/em\u003eCas12a (Schindele and Puchta \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) after \u003cem\u003eAsc\u003c/em\u003eI digestion. The pDe-Cas9, pDe-EC-tt\u003cem\u003eLb\u003c/em\u003eCas12a, and pEn-RZ-\u003cem\u003eLb\u003c/em\u003e-Chimera vectors were kindly provided by Dr. Holger Puchta (Karlsruhe Institute of Technology, Germany) (Schindele and Puchta \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The pDe-Cas9_NTPII was generated by inserting a synthetic NTPII cassette into the HindIII-digested pDe-Cas9 fragment using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA). The tt\u003cem\u003eLb\u003c/em\u003eCas12a fragment was purified by gel extraction using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) and inserted into the AscI-digested pDe-CAS9_NTPII vector using Ligation Mix (Takara, Shiga, Japan). Two complementary oligos containing a 24-bp CRISPR RNA (crRNA)-target sequence were annealed and cloned into the \u003cem\u003eBbs\u003c/em\u003eI-digested pEn-RZ-\u003cem\u003eLb\u003c/em\u003e-Chimera vector using Ligation Mix (Takara, Shiga, Japan). The crRNA is under the control of the Arabidopsis U6-26 promoter and flanked by the hammerhead and hepatitis delta virus ribozyme at the 5\u0026prime; and 3\u0026prime; sites, respectively. The crRNA cassette was confirmed by restriction enzyme analysis and sequencing. Finally, the crRNA cassette was transferred to the pDe-tt\u003cem\u003eLb\u003c/em\u003eCas12a_crRNA:SlGLK2_NTPII vector by the LR reaction using the Gateway LR Clonase II (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003ch3\u003eAgrobacterium-mediated transformation and isolation of mutants\u003c/h3\u003e\n\u003cp\u003eThe recombinant vector pDe-tt\u003cem\u003eLb\u003c/em\u003eCas12a_crRNA:SlGLK2_NTPII was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV2260 and transformed into tomato cv. Micro-Tom according to a previously described procedure (Sun et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In the T\u003csub\u003e0\u003c/sub\u003e generation, T-DNA positive lines were first isolated by polymerase chain reaction (PCR) of the tt\u003cem\u003eLb\u003c/em\u003eCas12a region in T-DNA using gene-specific primers. To confirm the presence of mutations in \u003cem\u003eSlGLK2\u003c/em\u003e, the sequence was confirmed using PCR amplification of the target region and subsequent direct sequencing of the PCR products. Transgene-negative homozygous alleles were verified in the T\u003csub\u003e1\u003c/sub\u003e generation using PCR-based sequencing.\u003c/p\u003e\n\u003ch3\u003eSequence analysis\u003c/h3\u003e\n\u003cp\u003eConserved domains and disordered regions were searched using InterProScan analysis (Quevillon et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Nuclear localization signal (NLS) prediction was conducted using LOCALIZER (Sperschneider et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and NLStradamus (Nguyen Ba et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). AlphaFold3 was used to predict the structure of SlGLK2 protein (Jumper et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSub-cloning of\u003c/b\u003e \u003cb\u003eSlGLK2\u003c/b\u003e \u003cb\u003egene and sequencing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from immature green fruit of Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele) and \u003cem\u003eCR-Slglk2-4\u003c/em\u003e mutant using an RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands). First-strand cDNA was synthesized using PrimeScript RT reagent (Takara, Shiga, Japan). The full-length coding sequences (CDSs) were amplified from the cDNA mix using KOD ONE (Toyobo, Tokyo, Japan) and gene-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To construct pENTR-SlGLK2(u) and pENTR-SlGLK2m, the amplified CDS fragments were cloned into a PCR-amplified pENTR vector using NEBuilder HiFi DNA Assembly Master Mix. The pENTR-SlGLKw construct, in which the functional \u003cem\u003eU\u003c/em\u003e allele of \u003cem\u003ethe SlGLK2\u003c/em\u003e gene was inserted, was generated by site-directed mutagenesis PCR using the pENTR-SlGLK2(u) plasmid as a template with gene-specific primers. The amplified fragment was then ligated using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA).\u003c/p\u003e\n\u003ch3\u003eSubcellular localization and transactivation assays of SlGLK2\u003c/h3\u003e\n\u003cp\u003eTo investigate the subcellular localization of wild-type and mutant SlGLK2 proteins, superfolder green fluorescence protein (sfGFP)-tagged expression vectors were constructed using the pGreenII62-SK backbone. First, a synthetic P19 silencing suppressor sequence was cloned into the \u003cem\u003eXba\u003c/em\u003eI and \u003cem\u003eKpn\u003c/em\u003eI sites of pGreenII62-SK (Hellens et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) to generate pGII62SK-P19. The P19 expression cassette was subsequently PCR-amplified and inserted into the \u003cem\u003eBamH\u003c/em\u003eI and \u003cem\u003eHind\u003c/em\u003eIII sites of the helper plasmid pSoup (Hellens et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) using the NEBuilder HiFi DNA Assembly Master Mix (NEB, MA, USA), resulting in pSoup-P19_K. This construct was introduced into the \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV3101 (MP90) by electroporation and used as the co-infiltration strain. The synthetic sfGFP gene was cloned into the pGreenII62-SK vector. The full-length coding sequences of \u003cem\u003eSlGLK2\u003c/em\u003e (Wid-type-type, \u003cem\u003eu\u003c/em\u003e allele mutant, 13-bp deletion allele mutant) were then PCR-amplified and inserted upstream of sfGFP in-frame, producing pGII62SK-SlGLK2\u003csup\u003eWT\u003c/sup\u003e:sfGFP, pGII62SK-SlGLK2\u003csup\u003e\u003cem\u003eu\u003c/em\u003e\u003c/sup\u003e:sfGFP, and pGII62SK-SlGLK2\u003csup\u003e\u003cem\u003eCR4\u003c/em\u003e\u003c/sup\u003e:sfGFP, respectively. These constructs were individually transformed into \u003cem\u003eAgrobacterium\u003c/em\u003e GV3101, which carried pSoup-P19_K.\u003c/p\u003e \u003cp\u003eAgro-infiltration was conducted according to a standard protocol (Hellens et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) as follows. The transformed \u003cem\u003eAgrobacterium\u003c/em\u003e strains were grown overnight, and 1 mL of the cultures was inoculated into 20 mL of Luria\u0026ndash;Bertani (LB) broth supplemented with 50 mg/L kanamycin, 50 mg/L rifampicin, 10 mg/L gentamicin, and 15 \u0026micro;M acetosyringone. The culture was incubated at 28\u0026deg;C until optical density at 600nm (OD\u003csub\u003e600\u003c/sub\u003e) reached\u0026thinsp;~\u0026thinsp;0.5. Subsequently, the grown cells were pelleted and resuspended in infiltration buffer (10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MES, and 150 \u0026micro;M acetosyringone, pH\u0026thinsp;=\u0026thinsp;5.6) to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0 and incubated at room temperature (RT) for 3 h in the dark. The suspensions were infiltrated into the abaxial side of the \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Then, plants were kept in the dark for 24 h at RT and then grown in a growth chamber for 48\u0026ndash;72 h. GFP fluorescence was observed after 4 d of incubation using a BX53 microscope (Evident, Tokyo, Japan) using a U-FBNA filter set.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of chlorophyll content\u003c/h2\u003e \u003cp\u003eImmature green fruit at 15 days after anthesis were harvested for total chlorophyll measurements, which were conducted according to a previous protocol (Ezura et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranscriptomic analysis\u003c/h3\u003e\n\u003cp\u003eThe fruit of the immature green stage at 15 d after anthesis was horizontally dissected into three parts: stem, middle, and tip ends. Then, pericarps from the stem end of WT and \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit and from the tip end of \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit were harvested and immediately frozen in liquid nitrogen. Total RNA was extracted using a Plant RNeasy Extraction Kit (Qiagen, Germany), according to the manufacturer\u0026rsquo;s instructions. Each triplicate sample contained pericarps from at least three independent fruit. To remove residual genomic DNA, RNA samples were further purified using the RNA Clean \u0026amp; Concentrator Kit-25 (Zymo Research, USA) with DNase I treatment. RNA quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). For RNA-seq analysis, mRNA was captured using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA), and sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer\u0026rsquo;s instructions. Paired-end sequencing (150 base pairs) for three replicates was performed using Illumina NovaSeq X Plus (Illumina, San Diego, CA, USA). Sequencing data were first cleaned using fastp ver. 0. 20.0 (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Cleaned reads were pseudo-aligned to transcripts from the Tomato ITAG4.0 genome using Salmon (v1.9.0) (Patro et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). After processing using tximport (Soneson et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), differentially expressed genes (DEGs) between WT and mutants (\u003cem\u003eCR-Slglk2-4\u003c/em\u003e) were identified using edgeR (Robinson et al., 2010) under iDEP 2.01 (Ge et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), applying a false discovery rate (FDR) cutoff of \u0026lt;\u0026thinsp;0.05. Gene Ontology (GO) term enrichment analysis was performed using ShinyGO v0.80 (Ge et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with a default setting (FDR cutoff\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for significance, relative to the whole-transcriptome background. For the comparison with the DEGs in the \u003cem\u003eSlGLK2\u003c/em\u003e-overexpression line (Nguyen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), DEGs in the \u003cem\u003eSlGLK2\u003c/em\u003e-overexpression line were re-analyzed using publicly-available data (SRA079879) through the same workflow.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from stem end and tip end of immature green fruits at 15 days after pollination of wild-type Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele) and \u003cem\u003eCR-Slglk2-4\u003c/em\u003e line using an RNeasy plant mini kit (Qiagen, Venlo, The Netherlands). Each biological replicate consisted of at least three fruits, and three biological replicates were analyzed. After DNase I treatment, genomic DNA contamination was checked by PCR. Reverse transcription was conducted using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was conducted on a StepOne Real-Time PCR System (Applied Biosystems, CA, USA) using THUNDERBIRD NEXT qPCR Mix (Toyobo, Osaka, Japan) according to the manufacturer\u0026rsquo;s instructions. \u003cem\u003eSlCAC\u003c/em\u003e (Solyc08g006960) was used as an internal control reference (Exp\u0026oacute;sito-Rodr\u0026iacute;guez et al. 2008). Primer sequences used in this experiment are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Relative expression levels were calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCT\u003c/sup\u003e method (Livak and Schmittgen 2001), with the Micro-Tom (\u003cem\u003eu\u003c/em\u003e allele) stem end sample set to 1.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene accession numbers\u003c/h2\u003e \u003cp\u003eAccession numbers for the genes used in this study are: SlGLK2 (Solyc10g008160), \u003cem\u003eSlPORB\u003c/em\u003e (Solyc10g006900), \u003cem\u003eSlMPEC\u003c/em\u003e (Solyc10g077040), \u003cem\u003eSlGGDR\u003c/em\u003e (Solyc03g115980), \u003cem\u003eSlCAB1C\u003c/em\u003e (Solyc02g071010), \u003cem\u003eSlLHCB2\u003c/em\u003e (Solyc07g047850), \u003cem\u003eSlLCYE\u003c/em\u003e (Solyc12g008980), and CONSTANS-like (Solyc05g009310).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGenome editing of\u003c/b\u003e \u003cb\u003euniform ripening\u003c/b\u003e \u003cb\u003e(\u003c/b\u003e\u003cb\u003eu\u003c/b\u003e\u003cb\u003e) allele by temperature-tolerant\u003c/b\u003e \u003cb\u003eLb\u003c/b\u003e\u003cb\u003eCas12a\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether genome editing could restore the function of a gene lost during tomato domestication, the function of the \u003cem\u003eSlGLK2\u003c/em\u003e gene was restored by re-editing the \u003cem\u003euniform ripening\u003c/em\u003e (\u003cem\u003eu\u003c/em\u003e) allele, which is widely used in cultivated tomato (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The \u003cem\u003eu\u003c/em\u003e-allele contains a single-base insertion in the coding region of \u003cem\u003eSlGLK2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The single-base insertion causes a frameshift and premature truncation of SlGLK2, resulting in the loss of its DNA-binding and dimerization domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). AlphaFold2 predictions of full-length SlGLK2 support that the insertion site is located within a disordered region (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This prompted us to create a compensatory frameshift near the insertion site and restore the full-length reading frame. The insertion site was located in an A/T-rich sequence, which was difficult to target with the Cas9 protein (even with the NG-Cas9 type Cas9, data not shown). Therefore, we employed the Cas12a system, which is suitable for targeting A/T-rich regions (Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, a mutated version of a \u003cem\u003eLachnospiraceae bacterium\u003c/em\u003e Cas12a, called temperature-tolerant \u003cem\u003eLb\u003c/em\u003eCas12a (tt\u003cem\u003eLb\u003c/em\u003eCas12a), which has enhanced activity at low temperatures (Schindele and Puchta \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) was used.\u003c/p\u003e \u003cp\u003eEditing with ttLbCas12a generated three T\u003csub\u003e0\u003c/sub\u003e lines carrying mutations near the target site. One of the three lines, \u003cem\u003eCR-Slglk2\u003c/em\u003e #2, displayed green shoulders at the stem end of the mature green (MG) fruit, resembling the \u003cem\u003eU\u003c/em\u003e allele (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nguyen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lupi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Direct sequencing of the T\u003csub\u003e0\u003c/sub\u003e lines #2 showed biallelic mutations in confirmed biallelic mutations, a 4-bp deletion (\u003cem\u003eCR-Slglk2-3\u003c/em\u003e allele), and a 13-bp deletion (\u003cem\u003eCR-Slglk2-4\u003c/em\u003e allele) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The \u003cem\u003eCR-Slglk2-4\u003c/em\u003e allele caused a frameshift that resulted in a normal reading frame, whereas the \u003cem\u003eCR-Slglk2-2\u003c/em\u003e allele caused a frameshift that resulted in another stop codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Other alleles lacking reading-frame restoration, a 3-bp deletion (\u003cem\u003eCR-Slglk2-1\u003c/em\u003e allele) or a 5-bp deletion (\u003cem\u003eCR-Slglk2-2\u003c/em\u003e allele), also produced fruit comparable to those of the original \u003cem\u003eu\u003c/em\u003e-allele plant.\u003c/p\u003e \u003cp\u003eTo confirm that the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e allele was responsible for the green shoulder phenotype, homozygous lines were obtained and analyzed in the T1 generation. The \u003cem\u003eCR-Slglk2-2/CR-Slglk2-2\u003c/em\u003e line produced fruit comparable to the original \u003cem\u003eu\u003c/em\u003e allele. In contrast, the \u003cem\u003eCR-Slglk2-4/CR-Slglk2-4\u003c/em\u003e line produced green shoulder fruit similar to that observed in a T\u003csub\u003e0\u003c/sub\u003e plant (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Chlorophyll content also increased in the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Together, these results indicate that the introduction of the 13-bp deletion into the \u003cem\u003eu\u003c/em\u003e allele successfully restored \u003cem\u003eSlGLK2\u003c/em\u003e function.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRestoration of SlGLK2 function in\u003c/b\u003e \u003cb\u003eCR-Slglk2-4\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eSlGLK2\u003c/em\u003e expression exhibits a latitudinal gradient from the stem to the stylar end of the immature green fruit in tomato (Nguyen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This latitudinal gradient was maintained in \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruits, with higher transcript levels at the stem end than at the stylar end (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Furthermore, \u003cem\u003eSlGLK2\u003c/em\u003e transcript levels were consistently higher in \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit than in WT at all examined positions, suggesting either an autoregulatory mechanism or a retrograde signal triggered by enhanced plastid development in the fruit. Thus, the restored green shoulder phenotype can be attributed to the recovery of protein function and elevated \u003cem\u003eSlGLK2\u003c/em\u003e expression, which maintains its spatial expression pattern. This result also confirmed that the \u003cem\u003euniform ripening\u003c/em\u003e phenotype of the \u003cem\u003eu\u003c/em\u003e allele results from the loss of SlGLK2 protein activity rather than an altered expression pattern.\u003c/p\u003e \u003cp\u003eTo further validate the restoration of SlGLK2 function, we determined that the mutation did not affect the splicing patterns. Full-length cDNA sequences were amplified from fruit-derived mRNA of wild-type and \u003cem\u003eCR-Slglk2-4\u003c/em\u003e and subsequently confirmed its sequence. According to the sequences and predicted protein sequences (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), other than the mutation site, the results indicated that the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e mutation had no impact on the overall splicing patterns.\u003c/p\u003e \u003cp\u003eGLK transcription factors are GARP transcription activators with a MYB-like DNA-binding domain, a conserved GOLDEN2 C-terminal (GCT) box, and a nuclear localization signal (NLS) sequence in the upstream region of the MYB-like DNA-binding domain (Hall et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Rossini et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Fitter et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The comparison of amino acid sequences and predicted structures showed that the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e mutation was located upstream of the MYB-like DNA-binding domain, in a region poorly conserved among plant orthologs (Fig. S3, S4). NLS predictions using LOCALIZER (Sperschneider et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and NLStradamus (Nguyen Ba et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) confirmed that the additional mutation did not alter the NLS signal upstream of the MYB-like domain (Fig. S5). Consistently, the sfGFP fusion proteins of SlGLK2\u003csup\u003eWT\u003c/sup\u003e and SlGLK2\u003csup\u003e\u003cem\u003eCR4\u003c/em\u003e\u003c/sup\u003e were localized to the nucleus, whereas no signal of SlGLK2\u003csup\u003e\u003cem\u003eu\u003c/em\u003e\u003c/sup\u003e was detected (Fig. S6). Collectively, these results indicate that the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e allele restored nuclear localization of its translated protein.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRestoration of SlGLK2-associated gene expression patterns in\u003c/b\u003e \u003cb\u003eCR-Slglk2-4\u003c/b\u003e \u003cb\u003efruits\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether SlGLK2\u003csup\u003e\u003cem\u003eCR4\u003c/em\u003e\u003c/sup\u003e in the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e line regulates a gene set similar to functional SlGLK2, transcriptome analysis was conducted using stem or tip end tissue from immature green fruits of \u003cem\u003eu\u003c/em\u003e allele and \u003cem\u003eCR-Slglk2-4\u003c/em\u003e plants (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e - S4). In comparison with the \u003cem\u003eu\u003c/em\u003e allele, 161 genes were upregulated, and 26 genes were downregulated in the stem end of \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Of the 161 upregulated genes, 62 (%) overlapped with differentially upregulated genes in the \u003cem\u003eSlGLK2\u003c/em\u003e-overexpression line (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, Table S5), based on reanalysis of published RNA-seq data (Nguyen et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), whereas only three genes were commonly downregulated (Table S6). Notably, 129 of the 161 genes (75.2%) exhibited higher expression in the stem end than tip end of \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit, suggesting their gradient expression pattern. Furthermore, 34 out of the 61 genes (53.8%) were identified as potential direct targets of SlGLK2 (Table S6), exhibiting significant binding signals in prior chromatin immunoprecipitation sequence (ChIP-seq) analysis (Tu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGene ontology analysis of the 62 commonly upregulated gene set showed overrepresentation of biological process and cellular componet terms involved in chloroplast- and photosynthesis-associated processes, including \u0026ldquo;chlorophyll biosynthetic process (GO:0015995)\u0026rdquo;, \u0026ldquo;photosynthesis, light harvesting in photosystem I (GO:0009768)\u0026rdquo;, \u0026ldquo;photosystem I reaction center (GO:0009538)\u0026rdquo;, and \u0026ldquo;chloroplast thylakoid lumen (GO:0009543) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003ed; Table S7). To further validate whether these transcriptional changes, we tested the expression of the commonly upregulated genes with SlGLK2-binding signal in the previous ChIP-seq analysis (Tu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Seven genes present in both datasets were selected for quantitative RT-PCR analysis, which confirmed their elevated expression in \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruits (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These genes included key components of chlorophyll biosynthesis and light-harvesting complexes, such as protochlorophyllide reductase B (\u003cem\u003eSlPORB\u003c/em\u003e), Mg-protoporphyrin IX monomethyl ester cyclase (\u003cem\u003eSlMPEC\u003c/em\u003e), geranylgeranyl diphosphate reductase (\u003cem\u003eSlGGDR\u003c/em\u003e), chlorophyll a-b binding protein 1C (\u003cem\u003eSlCAB1C\u003c/em\u003e), light-harvesting complex B2 (\u003cem\u003eSlLHCB2\u003c/em\u003e). In addition, \u003cem\u003elycopene epsilon-cyclase\u003c/em\u003e (\u003cem\u003eSlLCYE\u003c/em\u003e), recently proposed as a potential SlGLK2-regulated gene in the carotenoid pathway (Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), was aloso upregulated in \u003cem\u003eCR-Slglk2-4\u003c/em\u003e. Notably, one transcription factor, CONSTANS-like zinc finger protein (Solyc05g009310), also displayed elevated expression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the function of a domestication-associated non-functional \u003cem\u003eSlGLK2\u003c/em\u003e allele was restored using an SDN1-category genome-editing approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The introduction of a compensatory frameshift into the \u003cem\u003eu\u003c/em\u003e allele recovered SlGLK2 protein activity, reinstated the downstream transcriptional program for plastid biogenesis, and restored the green shoulder phenotype of the fruit. These results demonstrate that conventional SDN1-based genome editing can be used not only to disrupt genes to study their function but also to reactivate dormant genes that were lost during domestication and crop improvements.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe re-editing of domestication-derived, non-functional alleles as a strategy to reactivate dormant genes\u003c/h2\u003e \u003cp\u003eDomesticated plants unintentionally lose beneficial genes during domestication. Non-processed pseudogenes arise from duplication in genomic DNA and subsequent disablement, most commonly through disruptive frameshift mutations or premature stop codon formation (Akhunov et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although many pseudogenes have lost their function at the translational level due to a frameshift-associated premature stop codon and reduced or undetectable expression levels of their transcripts, a considerable number of pseudogenes are also transcribed (Pink et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sisu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In tomato, the protein function of the \u003cem\u003eSlGLK2\u003c/em\u003e gene was lost due to truncation of the translated protein by a premature stop codon, whereas the expression level was maintained (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Early tomato domestication and improvement have been focused on fruit appearance and yield rather than stress tolerance and fruit nutrition (Powell et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Bolger et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tieman et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For instance, widely-cultivated varieties produce large fruits but are substantially more sensitive to salt stress than their wild ancestors. A major variant, an in-frame, six-base insertion in the coding sequence of the \u003cem\u003eSlHAK20\u003c/em\u003e gene, which encodes a clade IV HAK/KUP/KT transporter, is associated with Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e homeostasis and mediates salt tolerance in tomato (Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The C-to-T substitution at nucleotide position 265 of \u003cem\u003eSlBBX18\u003c/em\u003e was selected during tomato evolution, increasing drought sensitivity due to premature stop codon formation (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, a deleterious mutation in the \u003cem\u003eSUPPRESSOR OF SP2\u003c/em\u003e (\u003cem\u003eSSP2\u003c/em\u003e) gene in cultivated tomato was repaired by base editing, resulting in early fruit yield (Glaus et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To our knowledge, this is the first report to show that restoring gene function lost during plant domestication or breeding can be achieved using simple deletion-type genome editing of the SDN1-category. In addition to the introduction of gain-of-function mutations targeting transcriptional regulatory regions (Rodr\u0026iacute;guez-Leal et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oliva et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), this approach may provide an alternative method for introducing attractive traits or enhancing target traits in cultivated varieties in a short period.\u003c/p\u003e \u003cp\u003eConventional breeding methods, which involve crossing with wild species to introduce the target locus, are susceptible to phenomena known as linkage drag and hitchhiking, where traits unrelated to the desired phenotypes are inadvertently introduced. This study demonstrates the practical use of genome editing to overcome these limitations. Future research should explore the integration of advanced genome-editing technologies, such as prime editing and CRISPR/Cas-based homologous recombination (HR), which offer precision and range beyond the capabilities of standard SDN1 techniques.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePutative downstream factors associated with SlGLK2-dependent transcriptional programs in tomato fruit\u003c/h2\u003e \u003cp\u003eThe GLK family functions as a central regulator of plastid development and coordinates a wide range of plastid-associated processes (Fitter et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Waters et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zubo et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hern\u0026aacute;ndez-Verdeja and Lundgren \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In tomato, chloroplasts differentiate into chromoplasts, accumulating health-promoting nutrients such as carotenoids (Sadali et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gong et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Enhancing plastid development in fruits is a promising approach for improving the nutritional quality of fruit-bearing crops (Wang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Alves et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the underlying processes of plastid development and differentiation, particularly their regulatory mechanisms, in developing fruits remain unclear.\u003c/p\u003e \u003cp\u003eOur comparative transcriptomic analysis identified 62 genes that were commonly upregulated in both \u003cem\u003eCR-Slglk2-4\u003c/em\u003e and \u003cem\u003eSlGLK2ox\u003c/em\u003e fruits, representing potential downstream targets of SlGLK2-regulated transcriptional pathway in developing fruit. Notably, 34 of these genes, including well-known photosynthesis-related genes and some regulatory factors, overlapped with previous ChIP-seq data (Tu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting potential direct regulation by SlGLK2.\u003c/p\u003e \u003cp\u003eAmong the commonly upregulated genes, we identified a CONSTANS-like zinc finger protein (Solyc05g009310), which is homologous to Arabidopsis Class III BBX subfamily members (BBX14/15/16/17) (Khanna et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In Arabidopsis, BBX16, which directly promotes the expression of SUPERROOT 2 (SUR2), which encodes a suppressor of auxin biosynthesis (Zhang et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), is directly regulated by GLK1 during seedling photomorphogenesis (Veciana et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence (Buelbuel et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Supporting this, the senescence-downregulated genes (SDGs), including the light-harvesting complex and PORB, were highly expressed in the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit. In tomato, CONSTANS-like transcription factors, such as SlCOL1 (Solyc02g089540) and SlBBX24 (Solyc06g073180), have been shown to stabilize SlGLK2 protein, thereby enhancing chlorophyll biosynthesis in fruits (Cui et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Considering that only a few transcriptional regulators were induced in \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit, Solyc05g009310 may contribute to plastid biogenesis secondary regulator SlGLK2-associated transcriptional regulation in tomato fruit, although its precise role remains to be determined. Future studies on isolated candidate genes will further elucidate the regulatory mechanisms underlying plastid biogenesis during tomato fruit development.\u003c/p\u003e \u003cp\u003eIn addition to downstream targets, \u003cem\u003eSlGLK2\u003c/em\u003e transcript level itself was also elevated in the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruit. Since \u003cem\u003eSlGLK2\u003c/em\u003e was not identified as a direct transcriptional target of SlGLK2 in a previous ChIP-seq analysis (Tu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), this increase is unlikely to result from direct autoregulation. Instead, it may reflect indirect positive feedback mediated by plastid-to-nucleus retrograde signaling. Consistent with this hypothesis, previous study in Arabidopsis have shown reduced \u003cem\u003eGLK1\u003c/em\u003e expression in \u003cem\u003eglk1\u003c/em\u003e/\u003cem\u003eglk2\u003c/em\u003e mutant under condition that promotes chloroplast development, suggesting the presence of some positive direct or indirect feedback regulation between GLK function and its own expression (Quevedo et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Considering this, the enhanced chloroplast biogenesis caused by restored GLK2 function might generate plastid-derived signals that secondarily promote nuclear \u003cem\u003eSlGLK2\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003eImportantly, \u003cem\u003eSlGLK2\u003c/em\u003e expression in \u003cem\u003eCR-Slglk2-4\u003c/em\u003e fruits exhibited a spatial pattern that closely paralleled chlorophyll accumulation along the stem-to-tip axis. This positional concordance supports a model in which local plastid status and retrograde signaling reinforce SlGLK2-regulated transcriptional programs in a position-dependent manner, thereby contributing to the pronounced green shoulder phenotype observed in this line.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCR-Slglk2-4\u003c/b\u003e \u003cb\u003eas a model resource for studying plastid biogenesis in tomato fruit\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe miniature cultivar Micro-Tom has been widely used as a model system for studying fruit physiology and development due to its small plant size, shorter life cycle, and extensive mutant resources (Carvalho et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Shikata and Ezura \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gasparini et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, Micro-Tom carries the \u003cem\u003eu\u003c/em\u003e allele (Carvalho et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which limits its utility for study of chloroplast development in fruit. In the \u003cem\u003eCR-Slglk2-4\u003c/em\u003e line, plastid development in fruit is restored within the genetic background of cultivated tomato, resulting in a pronounced green shoulder phenotype. Importantly, this restoration reflects functional recovery of SlGLK2 without introducing transgenes or large-scale genomic modifications. As \u003cem\u003eCR-Slglk2-4\u003c/em\u003e retains the advantageous characteristics of Micro-Tom while exhibiting spatially distinct plastid development within a single fruit, this line provides a useful genetic resource for investigating the regulatory mechanisms underlying plastid biogenesis and its spatial modulation during tomato fruit development.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Holger Putcha (Karlsruhe Institute of Technology, Germany) for the pDe-Cas9, pDe-EC-ttLbCas12a, and pEn-RZ-Lb-Chimera vectors. Seed of Micro-Tom NBRP-Japan line (TOMJPF00001) was obtained from the University of Tsukuba, Tsukuba Plant Innovation Research Center, through the National Bio-Resource Project (NBRP) of MEXT/AMED, Japan. We thank Dr. Kiyosada Kawai (Japan International Research Center for Agricultural Sciences, Japan) for kindly sharing Microscopy BX53 (Evident Scientific, Nagano, Japan). We thank all others who kindly shared the experimental tools and resources for this study. This work was financially supported by the Japan Science and Technology Agency (JST) (Grant No. 20J01560 and 24K17885 to K.E.). The author would like to thank Editage (www.editage.com) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnquiries about data and materials availability should be directed to the corresponding author. Raw transcriptomic data are deposited at GEO (accession number XXXX).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Japan Science and Technology Agency (JST) (Grant No. 20J01560 and 24K17885 to K.E.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author has no relevant financial or non-financial interests to disclose\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.E. conceptualized and designed the research. K.E. performed all experiments. K.E. wrote and revised the manuscript. K.E. acquired funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkhunov ED, Sehgal S, Liang H, et al (2013) Comparative analysis of syntenic genes in grass genomes reveals accelerated rates of gene structure and coding sequence evolution in polyploid wheat. Plant Physiol 161:252\u0026ndash;265. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.112.205161\u003c/span\u003e\u003cspan address=\"10.1104/pp.112.205161\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlves FRR, Lira BS, Pikart FC, et al (2020) Beyond the limits of photoperception: constitutively active PHYTOCHROME B2 overexpression as a means of improving fruit nutritional quality in tomato. Plant Biotechnol J 18:2027\u0026ndash;2041. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.13362\u003c/span\u003e\u003cspan address=\"10.1111/pbi.13362\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolger A, Scossa F, Bolger ME, et al (2014) The genome of the stress-tolerant wild tomato species Solanum pennellii. Nat Genet 46:1034\u0026ndash;1038. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ng.3046\u003c/span\u003e\u003cspan address=\"10.1038/ng.3046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuelbuel S, Sakuraba Y, Sedaghatmehr M, et al (2023) Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence. Plant J 116:251\u0026ndash;268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/tpj.16374\u003c/span\u003e\u003cspan address=\"10.1111/tpj.16374\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarvalho RF, Campos ML, Pino LE, et al (2011) Convergence of developmental mutants into a single tomato model system: \u0026ldquo;Micro-Tom\u0026rdquo; as an effective toolkit for plant development research. Plant Methods 7:18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1746-4811-7-18\u003c/span\u003e\u003cspan address=\"10.1186/1746-4811-7-18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen K, Wang Y, Zhang R, et al (2019) Crispr/cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol 70:667\u0026ndash;697. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-arplant-050718-100049\u003c/span\u003e\u003cspan address=\"10.1146/annurev-arplant-050718-100049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884\u0026ndash;i890. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/bty560\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/bty560\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui L, Zheng F, Zhang C, et al (2024) Tomato CONSTANS-LIKE SlCOL1 regulates the content of chlorophyll in fruit by stabilizing the GOLDEN2-LIKE protein. J Integr Agric. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jia.2024.11.022\u003c/span\u003e\u003cspan address=\"10.1016/j.jia.2024.11.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEzura K, Lu Y, Suzuki Y, et al (2024) Class II knotted-like homeodomain protein SlKN5 with BEL1-like homeodomain proteins suppresses fruit greening in tomato fruit. Plant J 118:2037\u0026ndash;2054. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/tpj.16727\u003c/span\u003e\u003cspan address=\"10.1111/tpj.16727\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEzura K, Nakamura A, Mitsuda N (2022) Genome-wide characterization of the TALE homeodomain family and the KNOX-BLH interaction network in tomato. Plant Mol Biol 109:799\u0026ndash;821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11103-022-01277-6\u003c/span\u003e\u003cspan address=\"10.1007/s11103-022-01277-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348\u0026ndash;359. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/tpj.12554\u003c/span\u003e\u003cspan address=\"10.1111/tpj.12554\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng Y, Wang Z, Chien K-Y, et al (2022) \u0026ldquo;Pseudo-pseudogenes\u0026rdquo; in bacterial genomes: Proteogenomics reveals a wide but low protein expression of pseudogenes in Salmonella enterica. Nucleic Acids Res 50:5158\u0026ndash;5170. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkac302\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkac302\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitter DW, Martin DJ, Copley MJ, et al (2002) GLK gene pairs regulate chloroplast development in diverse plant species. Plant J 31:713\u0026ndash;727. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-313x.2002.01390.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313x.2002.01390.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Gonda I, Sun H, et al (2019) The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor. Nat Genet 51:1044\u0026ndash;1051. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41588-019-0410-2\u003c/span\u003e\u003cspan address=\"10.1038/s41588-019-0410-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGasparini K, Figueiredo YG, de Aquino LM, et al (2025) Micro-Tom tomato: from ornamental horticulture to fundamental research. HORTIC ADV 3:8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s44281-025-00062-x\u003c/span\u003e\u003cspan address=\"10.1007/s44281-025-00062-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe SX, Jung D, Yao R (2020) ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics 36:2628\u0026ndash;2629. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btz931\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btz931\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe SX, Son EW, Yao R (2018) iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics 19:534. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12859-018-2486-6\u003c/span\u003e\u003cspan address=\"10.1186/s12859-018-2486-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlaus AN, Brechet M, Swinnen G, et al (2025) Repairing a deleterious domestication variant in a floral regulator gene of tomato by base editing. Nat Genet 57:231\u0026ndash;241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41588-024-02026-9\u003c/span\u003e\u003cspan address=\"10.1038/s41588-024-02026-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong J, Li Y, Shen X, et al (2024) Diversity in plastids contributes to variation in fruit color. Sci Hortic 337:113471. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2024.113471\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2024.113471\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall LN, Rossini L, Cribb L, Langdale JA (1998) GOLDEN 2: a novel transcriptional regulator of cellular differentiation in the maize leaf. Plant Cell 10:925\u0026ndash;936. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.10.6.925\u003c/span\u003e\u003cspan address=\"10.1105/tpc.10.6.925\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellens RP, Edwards EA, Leyland NR, et al (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819\u0026ndash;832. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/a:1006496308160\u003c/span\u003e\u003cspan address=\"10.1023/a:1006496308160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez-Verdeja T, Lundgren MR (2024) GOLDEN2‐LIKE transcription factors: A golden ticket to improve crops? Plants, People, Planet 6:79\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ppp3.10412\u003c/span\u003e\u003cspan address=\"10.1002/ppp3.10412\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJumper J, Evans R, Pritzel A, et al (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596:583\u0026ndash;589. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-021-03819-2\u003c/span\u003e\u003cspan address=\"10.1038/s41586-021-03819-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhanna R, Kronmiller B, Maszle DR, et al (2009) The Arabidopsis B-box zinc finger family. Plant Cell 21:3416\u0026ndash;3420. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.109.069088\u003c/span\u003e\u003cspan address=\"10.1105/tpc.109.069088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Ai G, Wang Y, et al (2024) A truncated B-box zinc finger transcription factor confers drought sensitivity in modern cultivated tomatoes. Nat Commun 15:8013. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-51699-7\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-51699-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Zhang Y, Xia L, Qi Y (2020) CRISPR-Cas12a enables efficient biallelic gene targeting in rice. Plant Biotechnol J 18:1351\u0026ndash;1353. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.13295\u003c/span\u003e\u003cspan address=\"10.1111/pbi.13295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Jian Y, Mao Y, et al (2022) \u0026ldquo;Omics\u0026rdquo; insights into plastid behavior toward improved carotenoid accumulation. Front Plant Sci 13:1001756. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2022.1001756\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2022.1001756\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLupi ACD, Lira BS, Gramegna G, et al (2019) Solanum lycopersicum GOLDEN 2-LIKE 2 transcription factor affects fruit quality in a light- and auxin-dependent manner. PLoS ONE 14:e0212224. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0212224\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0212224\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMenz J, Modrzejewski D, Hartung F, et al (2020) Genome edited crops touch the market: A view on the global development and regulatory environment. Front Plant Sci 11:586027. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2020.586027\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2020.586027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMesa T, Munn\u0026eacute;-Bosch S (2023) α-Tocopherol in chloroplasts: Nothing more than an antioxidant? Curr Opin Plant Biol 74:102400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pbi.2023.102400\u003c/span\u003e\u003cspan address=\"10.1016/j.pbi.2023.102400\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen Ba AN, Pogoutse A, Provart N, Moses AM (2009) NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinformatics 10:202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2105-10-202\u003c/span\u003e\u003cspan address=\"10.1186/1471-2105-10-202\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen CV, Vrebalov JT, Gapper NE, et al (2014) Tomato GOLDEN2-LIKE transcription factors reveal molecular gradients that function during fruit development and ripening. Plant Cell 26:585\u0026ndash;601. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.113.118794\u003c/span\u003e\u003cspan address=\"10.1105/tpc.113.118794\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNonaka S, Arai C, Takayama M, et al (2017) Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Sci Rep 7:7057. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-017-06400-y\u003c/span\u003e\u003cspan address=\"10.1038/s41598-017-06400-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliva R, Ji C, Atienza-Grande G, et al (2019) Broad-spectrum resistance to bacterial blight in rice using genome editing. Nat Biotechnol 37:1344\u0026ndash;1350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41587-019-0267-z\u003c/span\u003e\u003cspan address=\"10.1038/s41587-019-0267-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatro R, Duggal G, Love MI, et al (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417\u0026ndash;419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.4197\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.4197\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePink RC, Wicks K, Caley DP, et al (2011) Pseudogenes: pseudo-functional or key regulators in health and disease? RNA 17:792\u0026ndash;798. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1261/rna.2658311\u003c/span\u003e\u003cspan address=\"10.1261/rna.2658311\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePodevin N, Davies HV, Hartung F, et al (2013) Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding. Trends Biotechnol 31:375\u0026ndash;383. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tibtech.2013.03.004\u003c/span\u003e\u003cspan address=\"10.1016/j.tibtech.2013.03.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowell ALT, Nguyen CV, Hill T, et al (2012) Uniform ripening encodes a Golden 2-like transcription factor regulating tomato fruit chloroplast development. Science 336:1711\u0026ndash;1715. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1222218\u003c/span\u003e\u003cspan address=\"10.1126/science.1222218\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuevedo M, Kubalov\u0026aacute; I, Brun A, et al (2025) Retrograde signals control dynamic changes to the chromatin state at photosynthesis-associated loci. Nat Commun 16:6527. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-025-61831-w\u003c/span\u003e\u003cspan address=\"10.1038/s41467-025-61831-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuevillon E, Silventoinen V, Pillai S, et al (2005) InterProScan: protein domains identifier. Nucleic Acids Res 33:W116-20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gki442\u003c/span\u003e\u003cspan address=\"10.1093/nar/gki442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Leal D, Lemmon ZH, Man J, et al (2017) Engineering quantitative trait variation for crop improvement by genome editing. Cell 171:470\u0026ndash;480.e8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2017.08.030\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2017.08.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRossini L, Cribb L, Martin DJ, Langdale JA (2001) The maize golden2 gene defines a novel class of transcriptional regulators in plants. Plant Cell 13:1231\u0026ndash;1244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.13.5.1231\u003c/span\u003e\u003cspan address=\"10.1105/tpc.13.5.1231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadali NM, Sowden RG, Ling Q, Jarvis RP (2019) Differentiation of chromoplasts and other plastids in plants. Plant Cell Rep 38:803\u0026ndash;818. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00299-019-02420-2\u003c/span\u003e\u003cspan address=\"10.1007/s00299-019-02420-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindele P, Puchta H (2020) Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing. Plant Biotechnol J 18:1118\u0026ndash;1120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.13275\u003c/span\u003e\u003cspan address=\"10.1111/pbi.13275\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShikata M, Ezura H (2016) Micro-Tom Tomato as an Alternative Plant Model System: Mutant Collection and Efficient Transformation. Methods Mol Biol 1363:47\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-4939-3115-6_5\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4939-3115-6_5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh J, Gezan SA, Vallejos CE (2019) Developmental Pleiotropy Shaped the Roots of the Domesticated Common Bean (Phaseolus vulgaris). Plant Physiol 180:1467\u0026ndash;1479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.18.01509\u003c/span\u003e\u003cspan address=\"10.1104/pp.18.01509\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh J, van der Knaap E (2022) Unintended consequences of plant domestication. Plant Cell Physiol 63:1573\u0026ndash;1583. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/pcp/pcac083\u003c/span\u003e\u003cspan address=\"10.1093/pcp/pcac083\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSisu C, Muir P, Frankish A, et al (2020) Transcriptional activity and strain-specific history of mouse pseudogenes. Nat Commun 11:3695. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-020-17157-w\u003c/span\u003e\u003cspan address=\"10.1038/s41467-020-17157-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoneson C, Love MI, Robinson MD (2015) Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. [version 2; peer review: 2 approved]. F1000Res 4:1521. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12688/f1000research.7563.2\u003c/span\u003e\u003cspan address=\"10.12688/f1000research.7563.2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSperschneider J, Catanzariti A-M, DeBoer K, et al (2017) LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell. Sci Rep 7:44598. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep44598\u003c/span\u003e\u003cspan address=\"10.1038/srep44598\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun H-J, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426\u0026ndash;431. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/pcp/pci251\u003c/span\u003e\u003cspan address=\"10.1093/pcp/pci251\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun T, Hazra A, Lui A, et al (2025) GLKs directly regulate carotenoid biosynthesis via interacting with GBFs in plants. New Phytol 246:645\u0026ndash;665. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.20457\u003c/span\u003e\u003cspan address=\"10.1111/nph.20457\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanksley SD, McCouch SR (1997) Seed banks and molecular maps: unlocking genetic potential from the wild. Science 277:1063\u0026ndash;1066. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.277.5329.1063\u003c/span\u003e\u003cspan address=\"10.1126/science.277.5329.1063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTieman D, Zhu G, Resende MFR, et al (2017) A chemical genetic roadmap to improved tomato flavor. Science 355:391\u0026ndash;394. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.aal1556\u003c/span\u003e\u003cspan address=\"10.1126/science.aal1556\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomlinson L, Yang Y, Emenecker R, et al (2019) Using CRISPR/Cas9 genome editing in tomato to create a gibberellin-responsive dominant dwarf DELLA allele. Plant Biotechnol J 17:132\u0026ndash;140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.12952\u003c/span\u003e\u003cspan address=\"10.1111/pbi.12952\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTu X, Ren S, Shen W, et al (2022) Limited conservation in cross-species comparison of GLK transcription factor binding suggested wide-spread cistrome divergence. Nat Commun 13:7632. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-022-35438-4\u003c/span\u003e\u003cspan address=\"10.1038/s41467-022-35438-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeciana N, Mart\u0026iacute;n G, Leivar P, Monte E (2022) BBX16 mediates the repression of seedling photomorphogenesis downstream of the GUN1/GLK1 module during retrograde signalling. New Phytol 234:93\u0026ndash;106. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.17975\u003c/span\u003e\u003cspan address=\"10.1111/nph.17975\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang JY, Doudna JA (2023) CRISPR technology: A decade of genome editing is only the beginning. Science 379:eadd8643. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.add8643\u003c/span\u003e\u003cspan address=\"10.1126/science.add8643\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Liu J, Feng Y, et al (2008) Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4. Plant J 55:89\u0026ndash;103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-313X.2008.03489.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-313X.2008.03489.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, Zhang H, Zhu H (2019) CRISPR technology is revolutionizing the improvement of tomato and other fruit crops. Hortic Res 6:77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41438-019-0159-x\u003c/span\u003e\u003cspan address=\"10.1038/s41438-019-0159-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Aguirre L, Rodr\u0026iacute;guez-Leal D, et al (2021) Dissecting cis-regulatory control of quantitative trait variation in a plant stem cell circuit. Nat Plants 7:419\u0026ndash;427. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41477-021-00898-x\u003c/span\u003e\u003cspan address=\"10.1038/s41477-021-00898-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Hong Y, Zhu G, et al (2020) Loss of salt tolerance during tomato domestication conferred by variation in a Na+ /K+ transporter. EMBO J 39:e103256. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15252/embj.2019103256\u003c/span\u003e\u003cspan address=\"10.15252/embj.2019103256\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaters MT, Wang P, Korkaric M, et al (2009) GLK transcription factors coordinate expression of the photosynthetic apparatus in Arabidopsis. Plant Cell 21:1109\u0026ndash;1128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.108.065250\u003c/span\u003e\u003cspan address=\"10.1105/tpc.108.065250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Huai J, Shang F, et al (2017) A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis. Plant Physiol 174:2487\u0026ndash;2500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.17.00418\u003c/span\u003e\u003cspan address=\"10.1104/pp.17.00418\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Z, Kang X, Lor VS, et al (2018) Characterization of a semidominant dwarfing PROCERA allele identified in a screen for CRISPR/Cas9-induced suppressors of loss-of-function alleles. Plant Biotechnol J 17:319\u0026ndash;321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.13027\u003c/span\u003e\u003cspan address=\"10.1111/pbi.13027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZubo YO, Blakley IC, Franco-Zorrilla JM, et al (2018) Coordination of Chloroplast Development through the Action of the GNC and GLK Transcription Factor Families. Plant Physiol 178:130\u0026ndash;147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.18.00414\u003c/span\u003e\u003cspan address=\"10.1104/pp.18.00414\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"GOLDEN2-LIKE, Fruit development, Plastid, Tomato (Solanum lycopersicum), Genome editing","lastPublishedDoi":"10.21203/rs.3.rs-8821050/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8821050/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant domestication and improvement processes have inadvertently led to the loss of gene functions that contribute to crop quality. In widely-cultivated tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) varieties, a \u003cem\u003euniform ripening\u003c/em\u003e (\u003cem\u003eu\u003c/em\u003e) mutation, a loss-of-function allele of the \u003cem\u003eSolanum lycopersicum Golden2-like 2\u003c/em\u003e (\u003cem\u003eSlGLK2\u003c/em\u003e) gene, has been selected to improve fruit appearance. However, this selection is associated with reduced nutritional quality of the fruit, lowering the levels of sugars, carotenoids, and tocopherols due to impaired plastid development. In this study, the function of \u003cem\u003eSlGLK2\u003c/em\u003e was restored by introducing an additional mutation. A frameshift was introduced via genome editing using the temperature-tolerant \u003cem\u003eLachnospiraceae bacterium\u003c/em\u003e ND 2006 Cas12a (\u003cem\u003eLb\u003c/em\u003eCas12a) system. A 13-bp deletion in the linker region of the SlGLK2 protein in the \u003cem\u003eSlglk2-4\u003c/em\u003e line corrected the reading frame and led to enhanced plastid development in the basal part of the fruit. Biochemical and transcriptomic analyses confirmed the functional restoration of SlGLK2 in the \u003cem\u003eSlglk2-4\u003c/em\u003e line. This study provides a model Micro-Tom line for studying plastid biogenesis in tomato fruits and demonstrates the potential of genome editing to revive the latent functions of pseudogenized genes in modern crops, offering a new approach for recovering traits lost during domestication.\u003c/p\u003e","manuscriptTitle":"Reactivation of a loss-of-function SlGLK2 allele by frame-restoring genome editing in tomato","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 08:20:38","doi":"10.21203/rs.3.rs-8821050/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T17:34:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T14:34:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T15:29:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T10:06:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289578741540869872132447370794425808056","date":"2026-04-13T00:11:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6387094875831584207489028452297120167","date":"2026-04-12T06:03:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286156523157871021362850513082424706251","date":"2026-04-08T08:48:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T08:53:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-09T14:19:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-09T14:15:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-02-08T10:55:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"31dbc73f-cd97-4b8b-b8bd-1ca03de9022e","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T17:39:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 08:20:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8821050","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8821050","identity":"rs-8821050","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}