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.

SlRBP1 promotes translational efficiency via SleIF4A2 to maintain chloroplast function in tomato.

Many glycine-rich RNA-binding proteins (GR-RBPs) have critical functions in RNA processing and metabolism. Here, we describe a role for the tomato (Solanum lycopersicum) GR-RBP SlRBP1 in regulating mRNA translation. We found that SlRBP1 knockdown mutants (slrbp1) displayed reduced accumulation of total chlorophyll and impaired chloroplast ultrastructure. These phenotypes were accompanied by deregulation of the levels of numerous key transcripts associated with chloroplast functions in slrbp1. Furthermore, native RNA immunoprecipitation-sequencing (nRIP-seq) recovered 61 SlRBP1-associated RNAs, most of which are involved in photosynthesis. SlRBP1 binding to selected target RNAs was validated by nRIP-qPCR. Intriguingly, the accumulation of proteins encoded by SlRBP1-bound transcripts, but not the mRNAs themselves, was reduced in slrbp1 mutants. Polysome profiling followed by RT-qPCR assays indicated that the polysome occupancy of target RNAs was lower in slrbp1 plants than in wild-type. Furthermore, SlRBP1 interacted with the eukaryotic translation initiation factor SleIF4A2. Silencing of SlRBP1 significantly reduced SleIF4A2 binding to SlRBP1-target RNAs. Taking these observations together, we propose that SlRBP1 binds to and channels RNAs onto the SleIF4A2 translation initiation complex and promotes the translation of its target RNAs to regulate chloroplast functions.

Dicer-like2b suppresses the wiry leaf phenotype in tomato induced by tobacco mosaic virus.

Dicer-like (DCL) proteins are principal components of RNA silencing, a major defense mechanism against plant virus infections. However, their functions in suppressing virus-induced disease phenotypes remain largely unknown. Here, we identified a role for tomato (Solanum lycopersicum) DCL2b in regulating the wiry leaf phenotype during defense against tobacco mosaic virus (TMV). Knocking out SlyDCL2b promoted TMV accumulation in the leaf primordium, resulting in a wiry phenotype in distal leaves. Biochemical and bioinformatics analyses showed that 22-nt virus-derived small interfering RNAs (vsiRNAs) accumulated less abundantly in slydcl2b mutants than in wild-type plants, suggesting that SlyDCL2b-dependent 22-nt vsiRNAs are required to exclude virus from leaf primordia. Moreover, the wiry leaf phenotype was accompanied by upregulation of Auxin Response Factors (ARFs), resulting from a reduction in trans-acting siRNAs targeting ARFs (tasiARFs) in TMV-infected slydcl2b mutants. Loss of tasiARF production in the slydcl2b mutant was in turn caused by inhibition of miRNA390b function. Importantly, silencing SlyARF3 and SlyARF4 largely restored the wiry phenotype in TMV-infected slydcl2b mutants. Our work exemplifies the complex relationship between RNA viruses and the endogenous RNA silencing machinery, whereby SlyDCL2b protects the normal development of newly emerging organs by excluding virus from these regions and thus maintaining developmental silencing.

NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation.

The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

SlRIP1b is a global organellar RNA editing factor, required for normal fruit development in tomato plants.

RNA editing in plant organelles involves numerous C-U conversions, which often restore evolutionarily conserved codons and may generate new translation initiation and termination codons. These RNA maturation events rely on a subset of nuclear-encoded protein cofactors. Here, we provide evidence of the role of SlRIP1b on RNA editing of mitochondrial transcripts in tomato (Solanum lycopersicum) plants. SlRIP1b is a RIP/MORF protein that was originally identified as an interacting partner of the organellar editing factor SlORRM4. Mutants of SlRIP1b, obtained by CRISPR/Cas9 strategy, exhibited abnormal carpel development and grew into fruit with more locules. RNA-sequencing revealed that SlRIP1b affects the C-U editing of numerous mitochondrial pre-RNA transcripts and in particular altered RNA editing of various cytochrome c maturation (CCM)-related genes. The slrip1b mutants display increased H2 O2 and aberrant mitochondrial morphologies, which are associated with defects in cytochrome c biosynthesis and assembly of respiratory complex III. Taken together, our results indicate that SlRIP1b is a global editing factor that plays a key role in CCM and oxidative phosphorylation system biogenesis during fruit development in tomato plants. These data provide important insights into the molecular roles of organellar RNA editing factors during fruit development.

A tomato receptor-like cytoplasmic kinase, SlZRK1, acts as a negative regulator in wound-induced jasmonic acid accumulation and insect resistance.

Jasmonates accumulate rapidly and act as key regulators in response to mechanical wounding, but few studies have linked receptor-like cytoplasmic kinases (RLCKs) to wound-induced jasmonic acid (JA) signaling cascades. Here, we identified a novel wounding-induced RLCK-XII-2 subfamily member (SlZRK1) in tomato (Solanum lycopersicum) that was closely related to Arabidopsis HOPZ-ETI-DEFICIENT 1 (ZED1)-related kinases 1 based on phylogenetic analysis. SlZRK1 was targeted to the plasma membrane of tobacco mesophyll protoplasts as determined by transient co-expression with the plasma membrane marker mCherry-H+-ATPase. Catalytic residue sequence analysis and an in vitro kinase assay indicated that SlZRK1 may act as a pseudokinase. To further analyse the function of SlZRK1, we developed two stable knock-out mutants by CRISPR/Cas9. Loss of SlZRK1 significantly altered the expression of genes involved in JA biosynthesis, salicylic acid biosynthesis, and ethylene response. Furthermore, after mechanical wounding treatment, slzrk1 mutants increased transcription of early wound-inducible genes involved in JA biosynthesis and signaling. In addition, JA accumulation after wounding and plant resistance to herbivorous insects also were enhanced. Our findings expand plant regulatory networks in the wound-induced JA production by adding RLCKs as a new component in the wound signal transduction pathway.

Molecular and functional diversity of organelle RNA editing mediated by RNA recognition motif-containing protein ORRM4 in tomato.

Plant organellar RNA editing is a distinct type of post-transcriptional RNA modification that is critical for plant development. We showed previously that the RNA editing factor SlORRM4 is required for mitochondrial function and fruit ripening in tomato (Solanum lycopersicum). However, a comprehensive atlas of the RNA editing mediated by SlORRM4 is lacking. We observed that SlORRM4 is targeted to both chloroplasts and mitochondria, and its knockout results in pale-green leaves and delayed fruit ripening. Using high-throughput sequencing, we identified 12 chloroplast editing sites and 336 mitochondrial editing sites controlled by SlORRM4, accounting for 23% of chloroplast sites in leaves and 61% of mitochondrial sites in fruits, respectively. Analysis of native RNA immunoprecipitation sequencing revealed that SlORRM4 binds to 31 RNA targets; 19 of these targets contain SlORRM4-dependent editing sites. Large-scale analysis of putative SlORRM4-interacting proteins identified SlRIP1b, a RIP/MORF protein. Moreover, functional characterization demonstrated that SlRIP1b is involved in tomato fruit ripening. Our results indicate that SlORRM4 binds to RNA targets and interacts with SlRIP1b to broadly affect RNA editing in tomato organelles. These results provide insights into the molecular and functional diversity of RNA editing factors in higher plants.

Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis.

BACKGROUND: Superficial scald is a physiological disorder of apple fruit characterized by sunken, necrotic lesions appearing after prolonged cold storage, although initial injury occurs much earlier in the storage period. To determine the degree to which the transition to cell death is an active process and specific metabolism involved, untargeted metabolic and transcriptomic profiling was used to follow metabolism of peel tissue over 180 d of cold storage. RESULTS: The metabolome and transcriptome of peel destined to develop scald began to diverge from peel where scald was controlled using antioxidant (diphenylamine; DPA) or rendered insensitive to ethylene using 1-methylcyclopropene (1-MCP) beginning between 30 and 60 days of storage. Overall metabolic and transcriptomic shifts, representing multiple pathways and processes, occurred alongside α-farnesene oxidation and, later, methanol production alongside symptom development. CONCLUSIONS: Results indicate this form of peel necrosis is a product of an active metabolic transition involving multiple pathways triggered by chilling temperatures at cold storage inception rather than physical injury. Among multiple other pathways, enhanced methanol and methyl ester levels alongside upregulated pectin methylesterases are unique to peel that is developing scald symptoms similar to injury resulting from mechanical stress and herbivory in other plants.

Oligogalacturonic acids promote tomato fruit ripening through the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis at the transcriptional and post-translational levels.

BACKGROUND: Oligogalacturonic acids (OGs) are oligomers of alpha-1,4-linked galacturonosyl residues that are released from cell walls by the hydrolysis of polygalacturonic acids upon fruit ripening and under abiotic/biotic stress. OGs may induce ethylene production and fruit ripening, however, the mechanism(s) behind these processes is unknown. RESULTS: Tomato cultivar 'Ailsa Craig' (AC) and mutant Neverripe, ripening inhibitor, non-ripening, and colorless non-ripening fruits were treated with OGs at different stages. Only AC fruits at mature green stage 1 showed an advanced ripening phenomenon, although transient ethylene production was detected in all of the tomato fruits. Ethylene synthesis genes LeACS2 and LeACO1 were rapidly up-regulated, and the phosphorylated LeACS2 protein was detected after OGs treatment. Protein kinase/phosphatase inhibitors significantly affected the ripening process induced by the OGs. As a potential receptor of OGs, LeWAKL2 was also up-regulated in their presence. CONCLUSIONS: We demonstrated that OGs promoted tomato fruit ripening by inducing ethylene synthesis through the regulation of LeACS2 at transcriptional and post-translational levels.

[Research on the Tomato Metacaspase Protein Interactions with Ca2+ by Spectroscopy and Molecular Probe].

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Most of MCPs activation are the calcium dependent, but the mechanisms are still unknown. Based on the techniques of CD spectroscopy, fluorescence spectroscopy, and Terbium Stains-all probe, we selected three purified recombinant proteins from key residues mutated in tomato metacaspase (LeMCA1), including conserved catalytic site (C139A) mutant, N-sequenced cleaved site (K223G) mutant and the predicted Ca2+ binding sites (D116A/D117A) mutant, to explore the interaction mechanism of LeMCA1 and Ca2+. CD spectroscopy and Stains-all probe results suggested that the intense binding does not exist between LeMCA1 and Ca2+ as well as Ca2+ has little effect on the secondary structure of LeMCA1. However, fluorescence spectroscopy and Tb3+ probe results showed that Ca(2+)-induced the changes occur in the tertiary structure of LeMCA1, which contributes to the activation of zymogen. In addition, predicted Ca2+ binding residues, Asp-116 and Asp117, are the key sites resporisible for the Ca2+ interaction with LeMCA1, and the loss of these two residues resulted in decreased interaction. Our data firstly provided insight on the mechanism of the interaction between Ca2+ and recombinant purified Solanaceae type II metacaspase by spectroscopy and molecular probe techniques. Combined the results we got before from sequence-alignment and sites-mutation, the key residues Asp-116 and Asp117 affect the Ca(2+)-induced the changes of LeMCA1 tertiary structure. Our data provided information for the further biochemical and crystal assays of LeMCA1.

Techno-functional properties of active film based on guar gum-propolis and its application for "Nanguo" pears preservation.

Guar gum (GG) composite films, incorporating the ethanolic extract of propolis (EEP), were prepared and subjected to a comprehensive investigation of their functional characteristics. The addition of EEP resulted in a discernible enhancement in the opacity, moisture barrier capacity, and elongation at break. Incorporating EEP led to a noteworthy increase in the total phenolic and total flavonoid content of the films, resulting in superior antioxidant capacity upon GG-EEP films. Remarkably, the addition of 5 % EEP yielded noteworthy outcomes, manifesting in a DPPH radical scavenging rate of 47.60 % and the ABTS radical scavenging rate of 94.87 %, as well as FRAP and cupric reducing power of 331.98 mmol FeSO4-7H2O kg-1 and 56.95 µg TE mg-1, respectively. In addition, GG-EEP films demonstrated antifungal effect against Penicillium expansum and Aspergillus niger, along with a sustained antibacterial effect against Escherichia coli and Staphylococcus aureus. GG-EEP films had superior inhibitory ability against Gram-positive bacteria than Gram-negative bacteria. Crucially, GG-EEP composite films played a pivotal role in reducing both lesion diameter and depth, concurrently mitigating weight loss and firmness decline during the storage period of "Nanguo" pears. Therefore, GG-EEP composite films have the considerable potential to serve as advanced and effective active packaging materials for food preservation.

Fractionation and characterization of sodium carbonate-soluble fractions of cell wall pectic polysaccharides involved in the rapid mealiness of 'Hongjiangjun' apple fruit.

Apple flesh tends to turn mealy and textural deterioration commonly occurs during storage. The comparative investigation of three sub-fractions separated from sodium carbonate-soluble pectin (SSP) of 'Hongjiangjun' apples between crisp and mealy stages was performed to unveil the textural alterations related to mealiness. In situ immunofluorescence labelling showed that galactans declined in parenchyma cell walls during the fruit mealiness. FTIR analysis, monosaccharide compositions and structural polymers configurated that loss of rhammogalacturonan-I (RG-I) from SSP sub-fragments (SC0.0-P and S-M0.0-P) might be closely involved in the mealiness. The NMR spectroscopy revealed that loss of the substituted galactans from α-Rhap residues repeat unit in SC0.0-P constituting RG-I in crisp stage that subsequently converted to S-M0.0-P in mealy stage might be closely associated with the modifications of pectin in cell walls during mealiness. These findings provided novel evidence for understanding the underlying modifications of SSP polymers during the mealiness of 'Hongjiangjun' apples.

WALL-ASSOCIATED KINASE Like 14 regulates vascular tissue development in Arabidopsis and tomato.

Initiation of plant vascular tissue is regulated by transcriptional networks during development and in response to environmental stimuli. The WALL-ASSOCIATED KINASES (WAKs) and WAK-likes (WAKLs) are cell surface receptors involved in cell expansion and defence in cells with primary walls, yet their roles in regulation of vascular tissue development that contain secondary walls remains unclear. In this study, we showed tomato (Solanum lycopersicum) SlWAKL2 and the orthologous gene in Arabidopsis thaliana, AtWAKL14, were specifically expressed in vascular tissues. SlWAKL2-RNAi tomato plants displayed smaller fruit size with fewer seeds and vascular bundles compared to wild-type (WT) and over-expression (OE) lines. RNA-seq data showed that SlWAKL2-RNAi fruits down-regulated transcript levels of genes related to vascular tissue development compared to WT. Histological analysis showed T-DNA insertion mutant wakl14-1 had reduced plant stem length with fewer number of xylem vessels and interfascicular fibres compared to WT, with no significant differences in cellulose and lignin content. Mutant wakl14-1 also showed reduced number of vascular bundles in fruit. A proWAKL14::mCherry-WAKL14 fusion protein was able to complement wakl14-1 phenotypes and showed mCherry-WAKL14 associated with the plasma membrane. In vitro binding assays showed both SlWAKL2 and AtWAKL14 can interact with pectin and oligogalacturonides. Our results reveal novel roles of WAKLs in regulating vascular tissue development.

SRNAome parsing yields insights into tomato fruit ripening control.

Small RNAs have emerged as critical regulators in the expression and function of eukaryotic genomes at the post-transcriptional level. To elucidate the functions of microRNA (miRNAs) and endogenous small-interfering RNAs (siRNAs) in tomato fruit ripening process, the deep sequencing and bioinformatics methods were combined to parse the small RNAs landscape in three fruit-ripening stages (mature green, breaker and red-ripe) on a whole genome. Two species-specific miRNAs and two members of TAS3 family were identified, 590 putative phased small RNAs and 125 cis-natural antisense (nat-siRNAs) were also found in our results which enriched the tomato small RNAs repository and all of them showed differential expression patterns during fruit ripening. A large amount of the targets of the small RNAs were predicted to be involved in fruit ripening and ethylene pathway. Furthermore, the promoters of the conserved and novel miRNAs were found to contain the conserved motifs of TATA-box and CT microsatellites which were also found in Arabidopsis and rice, and several species-specific motifs were found in parallel.

Phytohormone ethylene mediates oligogalacturonic acid-induced growth inhibition in tomato etiolated seedlings.

Plant growth and immunity are tightly interconnected. Oligogalacturonic acids (OGs) are pectic fragments and have been well investigated in plant immunity as a damage-associated molecular pattern. However, little is known regarding how OGs affect plant growth. Here, we reveal that OGs inhibit the growth of intact etiolated seedling by using the horticultural crop tomato as a model. This inhibitory effect is partially suppressed by the action of ethylene biosynthesis inhibitors, or the gene silencing of SlACS2, an essential rate-limiting enzyme for ethylene biosynthesis, suggesting that SlACS2-mediated ethylene production promotes OG-induced growth inhibition. Furthermore, OGs treatment elevates the SlACS2 protein phosphorylation, and its decrease by the kinase inhibitor K252a partially rescue OG-induced growth inhibition, indicating that SlACS2 phosphorylation involves in OG-induced growth inhibition. Moreover, the mitogen-activated protein kinase SlMPK3 could be activated by OGs treatment and can directly phosphorylate SlACS2 in vitro, and the bimolecular fluorescence complementation combining with the yeast two-hybrid assay shows that SlMPK3 interacts with SlACS2, indicating that SlMPK3 may participate in modulating the OG-induced SlACS2 phosphorylation and growth inhibition. Our results reveal a regulatory mechanism at both the transcriptional and post-transcriptional levels by which OGs inhibit the growth of intact plant seedlings.

Comparative transcriptomic analysis provides novel insights into the difference in textural alteration between mealy and crisp apple patterns.

Mealiness is a common textural deterioration of several fruit after harvest. To unravel the underlying mechanism involved in mealiness, biochemical characterization and global transcriptomic profiling were comparatively performed between mealy 'Hongjiangjun' (HJJ) and crisp 'Fuji' apples. Sensory evaluation and SEM-based microstructure observation showed that HJJ apples appeared to be mealy in only 3 d at 23 ± 1 °C, while Fuji apples did not appear to be mealy even after 28 d of storage. Textural deterioration and ethylene burst occurred more sharply in HJJ apples than in Fuji apples during storage. The results obtained from the dimensional RNA-sequencing analysis showed that a much stronger upregulation of the transcription of genes encoding polygalacturonase (PG), pectin acetylesterase (PAE), pectinesterase (PE), ß-galactosidase (GAL), α-l-arabinofuranase (AF), and expansin (EXP) was observed in the pair of mealy HJJ apples vs. harvest than in the pair of Fuji apples after 28 d vs. harvest. The gene expression of ethylene responsive factor (ERF) was found to be strongly upregulated in HJJ apples compared with Fuji apples, which may mediate the regulation of downstream genes encoding cell wall-modifying enzymes. Weighted gene co-expression network analysis showed that the transcription factors MdbHLH63 and MdERF-like, and a constructure gene of MdGAL had strong connectivity with mealiness. Validation by qRT-PCR further confirmed the main findings obtained by RNA-sequencing. The occurrence of apple mealiness involves altered expression patterns of cell wall-modifying enzymes as well as MdbHLH63 and MdERF-like, which are core genes regulating the mealiness process. The above findings provide global insight into the difference in textural alteration between mealy and crisp apple patterns.

Chitinase III in pomegranate seeds (Punica granatum Linn.): a high-capacity calcium-binding protein in amyloplasts.

Chitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process.

Deciphering Precise Gene Transcriptional Expression Using gwINTACT in Tomato.

Functional gene transcription mainly occurs in the nucleus and has a significant role in plant physiology. The isolation of nuclei tagged in specific cell type (INTACT) technique provides an efficient and stable nucleus purification method to investigate the dynamic changes of nuclear gene transcriptional expression. However, the application of traditional INTACT in plants is still limited to seedlings or root cells because of severe chloroplast pollution. In this study, we proposed a newly designed and simplified INTACT based on mas-enhanced GFP (eGFP)-SlWIP2 (gwINTACT) for nuclear purification in tomato (Solanum lycopersicum) leaves, flowers, and fruits for the first time. The yield of the nucleus purified using gwINTACT from transgenic tomato leaves was doubled compared with using a traditional INTACT procedure, accompanied by more than 95% removal of chloroplasts. Relative gene expression of ethylene-related genes with ethylene treatment was reevaluated in gwINTACT leaves to reveal more different results from the traditional gene expression assay based on total RNA. Therefore, establishing the gwINTACT system in this study facilitates the precise deciphering of the transcriptional status in various tomato tissues, which lays the foundation for the further experimental study of nucleus-related molecular regulation on fruit ripening, such as ChIP-seq and ATAC-seq.

Glycine-rich RNA-binding cofactor RZ1AL is associated with tomato ripening and development.

Tomato ripening is a complex and dynamic process coordinated by many regulatory elements, including plant hormones, transcription factors, and numerous ripening-related RNAs and proteins. Although recent studies have shown that some RNA-binding proteins are involved in the regulation of the ripening process, understanding of how RNA-binding proteins affect fruit ripening is still limited. Here, we report the analysis of a glycine-rich RNA-binding protein, RZ1A-Like (RZ1AL), which plays an important role in tomato ripening, especially fruit coloring. To analyze the functions of RZ1AL in fruit development and ripening, we generated knockout cr-rz1al mutant lines via the CRISPR/Cas9 gene-editing system. Knockout of RZ1AL reduced fruit lycopene content and weight in the cr-rz1al mutant plants. RZ1AL encodes a nucleus-localized protein that is associated with Cajal-related bodies. RNA-seq data demonstrated that the expression levels of genes that encode several key enzymes associated with carotenoid biosynthesis and metabolism were notably downregulated in cr-rz1al fruits. Proteomic analysis revealed that the levels of various ribosomal subunit proteins were reduced. This could affect the translation of ripening-related proteins such as ZDS. Collectively, our findings demonstrate that RZ1AL may participate in the regulation of carotenoid biosynthesis and metabolism and affect tomato development and fruit ripening.