Rewiring of the Fruit Metabolome in Tomato Breeding.

Humans heavily rely on dozens of domesticated plant species that have been further improved through intensive breeding. To evaluate how breeding changed the tomato fruit metabolome, we have generated and analyzed a dataset encompassing genomes, transcriptomes, and metabolomes from hundreds of tomato genotypes. The combined results illustrate how breeding globally altered fruit metabolite content. Selection for alleles of genes associated with larger fruits altered metabolite profiles as a consequence of linkage with nearby genes. Selection of five major loci reduced the accumulation of anti-nutritional steroidal glycoalkaloids in ripened fruits, rendering the fruit more edible. Breeding for pink tomatoes modified the content of over 100 metabolites. The introgression of resistance genes from wild relatives in cultivars also resulted in major and unexpected metabolic changes. The study reveals a multi-omics view of the metabolic breeding history of tomato, as well as provides insights into metabolome-assisted breeding and plant biology.

Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing.

Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans-generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture. Our approach allows immediate selection and fixation of novel alleles in transgene-free plants and fine manipulation of yield components. Beyond a platform to enhance variation for diverse agricultural traits, our findings provide a foundation for dissecting complex relationships between gene-regulatory changes and control of quantitative traits.

Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation.

PIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Here, using synteny analysis, we show that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, we demonstrate that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. We provide further evidence that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Gibberellin and miRNA156-targeted SlSBP genes synergistically regulate tomato floral meristem determinacy and ovary patterning.

Many developmental processes associated with fruit development occur at the floral meristem (FM). Age-regulated microRNA156 (miR156) and gibberellins (GAs) interact to control flowering time, but their interplay in subsequent stages of reproductive development is poorly understood. Here, in tomato (Solanum lycopersicum), we show that GA and miR156-targeted SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL or SBP) genes interact in the tomato FM and ovary patterning. High GA responses or overexpression of miR156 (156OE), which leads to low expression levels of miR156-silenced SBP genes, resulted in enlarged FMs, ovary indeterminacy and fruits with increased locule number. Conversely, low GA responses reduced indeterminacy and locule number, and overexpression of a S. lycopersicum (Sl)SBP15 allele that is miR156 resistant (rSBP15) reduced FM size and locule number. GA responses were partially required for the defects observed in 156OE and rSBP15 fruits. Transcriptome analysis and genetic interactions revealed shared and divergent functions of miR156-targeted SlSBP genes, PROCERA/DELLA and the classical WUSCHEL/CLAVATA pathway, which has been previously associated with meristem size and determinacy. Our findings reveal that the miR156/SlSBP/GA regulatory module is deployed differently depending on developmental stage and create novel opportunities to fine-tune aspects of fruit development that have been important for tomato domestication.

Species coexistence by wide constant size spacing.

We consider the distribution of fruit pigeons of the genera Ptilinopus and Ducula on the island of New Guinea. Of the 21 species, between six and eight coexist inside humid lowland forests. We conducted or analyzed 31 surveys at 16 different sites, resurveying some sites in different years. The species coexisting at any single site in a single year are a highly nonrandom selection of the species to which that site is geographically accessible. Their sizes are both much more widely spread and more uniformly spaced than in random sets of species drawn from the locally available species pool. We also present a detailed case study of a highly mobile species that has been recorded on every ornithologically explored island in the West Papuan island group west of New Guinea. That species' rareness on just three well-surveyed islands within the group cannot be due to an inability to reach them. Instead, its local status decreases from abundant resident to rare vagrant in parallel with increasing weight proximity of the other resident species.

The ethylene-responsive transcription factor ERF024 is a novel regulator of climacteric fruit ripening in melon.

Fruit ripening is an essential developmental stage in Angiosperms triggered by hormonal signals such as ethylene, a major player in climacteric ripening. Melon is a unique crop showing both climacteric and non-climacteric cultivars, offering an ideal model for dissecting the genetic mechanisms underpinning this process. The major quantitative trait locus ETHQV8.1 was previously identified as a key regulator of melon fruit ripening. Here, we narrowed down ETHQV8.1 to a precise genomic region containing a single gene, the transcription factor CmERF024. Functional validation using CRISPR/Cas9 knock-out plants unequivocally identified CmERF024 as the causal gene governing ETHQV8.1. The erf024 mutants exhibited suppression of ethylene production, leading to a significant delay and attenuation of fruit ripening. Integrative multi-omic analyses encompassing RNA-seq, DAP-seq, and DNase-seq revealed the association of CmERF024 with chromatin accessibility and gene expression dynamics throughout fruit ripening. Our data suggest CmERF024 as a novel regulator of climacteric fruit ripening in melon.

Ploidy-specific transcriptomes shed light on the heterogeneous identity and metabolism of developing tomato pericarp cells.

Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication plays pivotal roles in plant growth and development and is associated with the activation of specific transcriptional programmes that are characteristic of each cell type, thereby defining their identity. In plants, endoreduplication is found in numerous organs and cell types, especially in agronomically valuable ones, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. We used the tomato pericarp tissue as a model system to explore the transcriptomes associated with endoreduplication progression during fruit growth. We confirmed that expression globally scales with ploidy level and identified sets of differentially expressed genes presenting only developmental-specific, only ploidy-specific expression patterns or profiles resulting from an additive effect of ploidy and development. When comparing ploidy levels at a specific developmental stage, we found that non-endoreduplicated cells are defined by cell division state and cuticle synthesis while endoreduplicated cells are mainly defined by their metabolic activity changing rapidly over time. By combining this dataset with publicly available spatiotemporal pericarp expression data, we proposed a map describing the distribution of ploidy levels within the pericarp. These transcriptome-based predictions were validated by quantifying ploidy levels within the pericarp tissue. This in situ ploidy quantification revealed the dynamic progression of endoreduplication and its cell layer specificity during early fruit development. In summary, the study sheds light on the complex relationship between endoreduplication, cell differentiation and gene expression patterns in the tomato pericarp.

SlMYC2 promotes SlLBD40-mediated cell expansion in tomato fruit development.

As a plant-specific transcription factor, lateral organ boundaries domain (LBD) protein was reported to regulate plant growth and stress response, but the functional research of subfamily II genes is limited. SlMYC2, a master regulator of Jasmonic acid response, has been found to exhibit high expression levels in fruit and has been implicated in the regulation of fruit ripening and resistance to Botrytis. However, its role in fruit expansion remains unknown. In this study, we present evidence that a subfamily II member of LBD, namely SlLBD40, collaborates with SlMYC2 in the regulation of fruit expansion. Overexpression of SlLBD40 significantly promoted fruit growth by promoting mesocarp cell expansion, while knockout of SlLBD40 showed the opposite result. Similarly, SlMYC2 knockout resulted in a significant decrease in cell expansion within the fruit. Genetic analysis indicated that SlLBD40-mediated cell expansion depends on the expression of SlMYC2. SlLBD40 bound to the promoter of SlEXPA5, an expansin gene, but did not activate its expression directly. While, the co-expression of SlMYC2 and SlLBD40 significantly stimulated the activation of SlEXPA5, leading to an increase in fruit size. SlLBD40 interacted with SlMYC2 and enhanced the stability and abundance of SlMYC2. Furthermore, SlMYC2 directly targeted and activated the expression of SlLBD40, which is essential for SlLBD40-mediated fruit expansion. In summary, our research elucidates the role of the interaction between SlLBD40 and SlMYC2 in promoting cell expansion in tomato fruits, thus providing novel insights into the molecular genetics underlying fruit growth.

The kiwifruit amyloplast proteome (kfALP): a resource to better understand the mechanisms underlying amyloplast biogenesis and differentiation.

The biogenesis and differentiation (B&D) of amyloplasts contributes to fruit flavor and color. Here, remodeling of starch granules, thylakoids and plastoglobules was observed during development and ripening in two kiwifruit (Actinidia spp.) cultivars - yellow-fleshed 'Hort16A' and green-fleshed 'Hayward'. A protocol was developed to purify starch-containing plastids with a high degree of intactness, and amyloplast B&D was studied using label-free-based quantitative proteomic analyses in both cultivars. Over 3000 amyloplast-localized proteins were identified, of which >98% were quantified and defined as the kfALP (kiwifruit amyloplast proteome). The kfALP data were validated by Tandem-Mass-Tag (TMT) labeled proteomics in 'Hort16A'. Analysis of the proteomic data across development and ripening revealed: 1) a conserved increase in the abundance of proteins participating in starch synthesis/degradation during both amyloplast B&D; 2) up-regulation of proteins for chlorophyll degradation and of plastoglobule-localized proteins associated with chloroplast breakdown and plastoglobule formation during amyloplast differentiation; 3) constitutive expression of proteins involved in ATP supply and protein import during amyloplast B&D. Interestingly, two different pathways of amyloplast B&D were observed in the two cultivars. In 'Hayward', significant increases in abundance of photosynthetic- and tetrapyrrole metabolism-related proteins were observed, but the opposite trend was observed in 'Hort16A'. In conclusion, analysis of the kfALP provides new insights into the potential mechanisms underlying amyloplast B&D with relevance to key fruit quality traits in contrasting kiwifruit cultivars.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

The Ca2+-MdCRF4-MdWRKY9 module negatively affects apple fruit watercore formation by suppressing the transcription of MdSOT2.

Watercore is a common physiological disease of Rosaceae plants, such as apples (Malus domestica), usually occurring during fruit ripening. Apple fruit with watercore symptoms is prone to browning and rotting, thus losing commercial viability. Sorbitol and calcium ions are considered key factors affecting watercore occurrence in apples. However, the mechanism by which they affect the occurrence of watercore remains unclear. Here, we identified that the transcription factor MdWRKY9 directly binds to the promoter of MdSOT2, positively regulates the transcription of MdSOT2, increases sorbitol content in fruit, and promotes watercore occurrence. Additionally, MdCRF4 can directly bind to MdWRKY9 and MdSOT2 promoters, positively regulating their expression. Since calcium ions can induce the ubiquitination and degradation of the transcription factor MdCRF4, they can inhibit the transcription of MdWRKY9 and MdSOT2 by degrading MdCRF4, thereby reducing the sorbitol content in fruit and inhibiting the occurrence of fruit watercore disease. Our data sheds light on how calcium ions mitigate watercore in fruit, providing molecular-level insights to enhance fruit quality artificially.

Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation.

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.

The MdCBF1/2-MdTST1/2 module regulates sugar accumulation in response to low temperature in apple.

Soluble sugar content is a key component in controlling fruit flavor, and its accumulation in fruit is largely determined by sugar metabolism and transportation. When the diurnal temperature range is greater, the fleshy fruits accumulated more soluble sugars and become more sweeter. However, the molecular mechanism underlying this response remains largely unknown. In this study, we verified that low-temperature treatment promoted soluble sugar accumulation in apple fruit and found that this was due to the upregulation of the Tonoplast Sugar Transporter genes MdTST1/2. A combined strategy using assay for transposase-accessible chromatin (ATAC) sequencing and gene expression and cis-acting elements analyses, we identified two C-repeat Binding Factors, MdCBF1 and MdCBF2, that were induced by low temperature and that might be upstream transcription factors of MdTST1/2. Further studies established that MdCBF1/2 could bind to the promoters of MdTST1/2 and activate their expression. Overexpression of MdCBF1 or MdCBF2 in apple calli and fruit significantly upregulated MdTST1/2 expression and increased the concentrations of glucose, fructose, and sucrose. Suppression of MdTST1 and/or MdTST2 in an MdCBF1/2-overexpression background abolished the positive effect of MdCBF1/2 on sugar accumulation. In addition, simultaneous silencing of MdCBF1/2 downregulated MdTST1/2 expression and apple fruits failed to accumulate more sugars under low-temperature conditions, indicating that MdCBF1/2-mediated sugar accumulation was dependent on MdTST1/2 expression. Hence, we concluded that the MdCBF1/2-MdTST1/2 module is crucial for sugar accumulation in apples in response to low temperatures. Our findings provide mechanistic components coordinating the relationship between low temperature and sugar accumulation as well as new avenues to improve fruit quality.

Class II knotted-like homeodomain protein SlKN5 with BEL1-like homeodomain proteins suppresses fruit greening in tomato fruit.

Knotted1-like homeodomain (KNOX) proteins are essential in regulating plant organ differentiation. Land plants, including tomato (Solanum lycopersicum), have two classes of the KNOX protein family, namely, class I (KNOX I) and class II KNOX (KNOX II). While tomato KNOX I proteins are known to stimulate chloroplast development in fruit, affecting fruit coloration, the role of KNOX II proteins in this context remains unclear. In this study, we employ CRISPR/Cas9 to generate knockout mutants of the KNOX II member, SlKN5. These mutants display increased leaf complexity, a phenotype commonly associated with reduced KNOX II activity, as well as enhanced accumulation of chloroplasts and chlorophylls in smaller cells within young, unripe fruit. RNA-seq data analyses indicate that SlKN5 suppresses the transcriptions of genes involved in chloroplast biogenesis, chlorophyll biosynthesis, and gibberellin catabolism. Furthermore, protein-protein interaction assays reveal that SlKN5 physically interacts with three transcriptional repressors from the BLH1-clade of BEL1-like homeodomain (BLH) protein family, SlBLH4, SlBLH5, and SlBLH7, with SlBLH7 showing the strongest interaction. CRISPR/Cas9-mediated knockout of these SlBLH genes confirmed their overlapping roles in suppressing chloroplast biogenesis, chlorophyll biosynthesis, and lycopene cyclization. Transient assays further demonstrate that the SlKN5-SlBLH7 interaction enhances binding capacity to regulatory regions of key chloroplast- and chlorophyll-related genes, including SlAPRR2-like1, SlCAB-1C, and SlGUN4. Collectively, our findings elucidate that the KNOX II SlKN5-SlBLH regulatory modules serve to inhibit fruit greening and subsequently promote lycopene accumulation, thereby fine-tuning the color transition from immature green fruit to mature red fruit.

Abscisic acid controls sugar accumulation essential to strawberry fruit ripening via the FaRIPK1-FaTCP7-FaSTP13/FaSPT module.

Strawberry is considered as a model plant for studying the ripening of abscisic acid (ABA)-regulated non-climacteric fruits, a process in which sugar plays a fundamental role, while how ABA regulates sugar accumulation remains unclear. This study provides a direct line of physiological, biochemical, and molecular evidence that ABA signaling regulates sugar accumulation via the FaRIPK1-FaTCP7-FaSTP13/FaSPT signaling pathway. Herein, FaRIPK1, a red-initial protein kinase 1 previously identified in strawberry fruit, not only interacted with the transcription factor FaTCP7 (TEOSINTE BRANCHEN 1, CYCLOIDEA, and PCF) but also phosphorylated the critical Ser89 and Thr93 sites of FaTCP7, which negatively regulated strawberry fruit ripening, as evidenced by the transient overexpression (OE) and virus-induced gene silencing transgenic system. Furthermore, the DAP-seq experiments revealed that FvTCP7 bound the motif "GTGGNNCCCNC" in the promoters of two sugar transporter genes, FaSTP13 (sugar transport protein 13) and FaSPT (sugar phosphate/phosphate translocator), inhibiting their transcription activities as determined by the electrophoretic mobility shift assay, yeast one-hybrid, and dual-luciferase reporter assays. The downregulated FaSTP13 and FaSPT transcripts in the FaTCP7-OE fruit resulted in a reduction in soluble sugar content. Consistently, the yeast absorption test revealed that the two transporters had hexose transport activity. Especially, the phosphorylation-inhibited binding of FaTCP7 to the promoters of FaSTP13 and FaSPT could result in the release of their transcriptional activities. In addition, the phosphomimetic form FaTCP7S89D or FaTCP7T93D could rescue the phenotype of FaTCP7-OE fruits. Importantly, exogenous ABA treatment enhanced the FaRIPK1-FaTCP7 interaction. Overall, we found direct evidence that ABA signaling controls sugar accumulation during strawberry fruit ripening via the "FaRIPK1-FaTCP7-FaSTP13/FaSPT" module.

Gynoecium and fruit development in Arabidopsis.

Flowering plants produce flowers and one of the most complex floral structures is the pistil or the gynoecium. All the floral organs differentiate from the floral meristem. Various reviews exist on molecular mechanisms controlling reproductive development, but most focus on a short time window and there has been no recent review on the complete developmental time frame of gynoecium and fruit formation. Here, we highlight recent discoveries, including the players, interactions and mechanisms that govern gynoecium and fruit development in Arabidopsis. We also present the currently known gene regulatory networks from gynoecium initiation until fruit maturation.

Histone H3K27 Demethylase SlJMJ3 Modulates Fruit Ripening in Tomato.

The histone lysine (K) demethylase 4 (KDM4/JHDM3) subfamily of jumonji domain-containing demethylases (JMJs) has been implicated in various aspects of plant development. However, their involvement in regulating the ripening of fleshy fruits remains unclear. Here, we identified SlJMJ3, a member of the KDM4/JHDM3 family, as a H3K27me3 demethylase in tomato (Solanum lycopersicum) that plays an important role in fruit ripening regulation. Overexpression of SlJMJ3 led to accelerated fruit ripening, whereas loss-of-function of SlJMJ3 delayed this process. Furthermore, we determined that SlJMJ3 exerts its regulatory function by modulating the expression of multiple ripening-related genes involved in ethylene biosynthesis and response, carotenoid metabolism, cell wall modification, transcriptional control, and DNA methylation modification. SlJMJ3 bound directly to the promoters of ripening-related genes harboring the CTCTGYTY motif and activates their expression. Additionally, SlJMJ3 reduced the levels of H3K27me3 at its target genes, thereby up-regulating their expression. In summary, our findings highlight the role of SlJMJ3 in the regulation of fruit ripening in tomato. By removing the methyl group from trimethylated histone H3 lysine 27 at ripening-related genes, SlJMJ3 acts as an epigenetic regulator that orchestrates the complex molecular processes underlying fruit ripening.

RNA editing factor SlORRM2 regulates the formation of fruit pointed tips in tomato.

Domestication of tomato (Solanum lycopersicum) has led to large variation in fruit size and morphology. The development of the distal end of the fruit is a critical factor in determining its overall shape. However, the intricate mechanisms underlying distal fruit development require further exploration. This study aimed to investigate the regulatory role of an organelle RNA recognition motif (RRM)-containing protein SlORRM2 in tomato fruit morphology development. Mutant plants lacking SlORRM2 exhibited fruits with pointed tips at the distal end. However, this phenotype could be successfully restored through the implementation of a "functional complementation" strategy. Our findings suggest that the formation of pointed tips in the fruits of the CR-slorrm2 mutants is linked to alterations in the development of the ovary and style. We observed a substantial decrease in the levels of indole-3-acetic acid (IAA) and altered expression of IAA-related response genes in the ovary and style tissues of CR-slorrm2. Moreover, our data demonstrated that SlORRM2 plays a role in regulating mitochondrial RNA editing sites, particularly within genes encoding various respiratory chain subunits. Additionally, the CR-slorrm2 mutants exhibited modified organellar morphology and increased levels of reactive oxygen species (ROS). These findings provide valuable insights into the mechanisms underlying the formation of fruit pointed tips in tomato and offer genetic resources for tomato breeding.

Persulfidation and phosphorylation of transcription factor SlWRKY6 differentially regulate tomato fruit ripening.

Cysteine desulfhydrase (LCD) catalyzes the generation of the signaling molecule hydrogen sulfide (H2S) in plants. In this study, we found that H2S can inhibit tomato (Solanum lycopersicum) fruit ripening and SlWRKY6 undergoes differential protein persulfidation in SlLCD1-overexpressing leaves. Then, further study indicated that SlWRKY6 could be persulfidated by H2S at Cys396. By construction of slwrky6 mutants and SlWRKY6-OE lines, we found that SlWRKY6 positively regulates leaf senescence and fruit ripening by activating the transcription of ripening-related genes STAYGREEN 1 (SlSGR1) and Senescence-Associated Gene 12 (SlSAG12). In addition, SlWRKY6 interacted with kinase SlMAPK4 and was phosphorylated at Ser33. Dual luciferase transient expression assays and electrophoretic mobility shift assays indicated that SlWRKY6 persulfidation attenuated its transcriptional regulation of target genes SlSGR1 and SlSAG12, whereas SlWRKY6 phosphorylation by SlMAPK4 activated the transcription of target genes to promote fruit ripening. Moreover, we provided evidence that SlWRKY6 persulfidation attenuated its SlMAPK4-mediated phosphorylation to inhibit tomato fruit ripening. By transient expression of SlWRKY6, SlWRKY6C396A, SlWRKY6S33A and SlWRKY6S33D in slwrky6 fruits, we found that SlWRKY6 persulfidation attenuated the expression of SlSGR1 and SlSAG12 thereby delaying tomato fruit ripening, while SlWRKY6 phosphorylation increased the expression of target genes. As tomato fruits ripened, endogenous H2S production decreased, while SlMAPK4 expression increased. Therefore, our findings reveal a model in which SlWRKY6 persulfidation due to higher endogenous H2S levels in un-ripened fruit inhibits its ability to activate SlSGR1 and SlSAG12 expression, while SlWRKY6 phosphorylation by SlMAPK4 activates its transcriptional activity, thereby promoting tomato fruit ripening.