Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template. The average mutation frequency (72.73%) and low replacement efficiency (7.69%) were achieved in T₀ transgenic plants respectively. None of homozygous mutation was detected in T₀ transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T₁ generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T₁ generations were acquired and further confirmed by genotype and phenotype characterization, with highlight of excellent storage performance, thus the recessive homozygous breeding elites with the character of long-shelf life were generated. Our results support that CRISPR/Cas9-induced gene replacement via HDR provides a valuable method for breeding elite innovation in tomato.

As global soil salinization continues to intensify, there is a need to enhance salt tolerance in crops. Understanding the molecular mechanisms of tomato (Solanum lycopersicum) roots’ adaptation to salt stress is of great significance to enhance its salt tolerance and promote its planting in saline soils. A combined analysis of the metabolome and transcriptome of S. lycopersicum roots under different periods of salt stress according to changes in phenotypic and root physiological indices revealed that different accumulated metabolites and differentially expressed genes (DEGs) associated with phenylpropanoid biosynthesis were significantly altered. The levels of phenylpropanoids increased and showed a dynamic trend with the duration of salt stress. Ferulic acid (FA) and spermidine (Spd) levels were substantially up-regulated at the initial and mid-late stages of salt stress, respectively, and were significantly correlated with the expression of the corresponding synthetic genes. The results of canonical correlation analysis screening of highly correlated DEGs and construction of regulatory relationship networks with transcription factors (TFs) for FA and Spd, respectively, showed that the obtained target genes were regulated by most of the TFs, and TFs such as MYB, Dof, BPC, GRAS, and AP2/ERF might contribute to the regulation of FA and Spd content levels. Ultimately, FA and Spd attenuated the harm caused by salt stress in S. lycopersicum, and they may be key regulators of its salt tolerance. These findings uncover the dynamics and possible molecular mechanisms of phenylpropanoids during different salt stress periods, providing a basis for future studies and crop improvement.

Background: Fruit cracking occurs easily under unsuitable environmental conditions and is one of the main types of damage that occurs in fruit production. It is widely accepted that plants have developed defence mechanisms and regulatory networks that respond to abiotic stress, which involves perceiving, integrating and responding to stress signals by modulating the expression of related genes. Fruit cracking is also a physiological disease caused by abiotic stress. It has been reported that a single or several genes may regulate fruit cracking. However, almost none of these reports have involved cracking regulatory networks.Results: Here, RNA expression in 0 h, 8 h and 30 h saturated irrigation-treated fruits from two contrasting tomato genotypes, ‘LA1698’ (cracking-resistant, CR) and ‘LA2683’ (cracking-susceptible, CS), was analysed by mRNA and lncRNA sequencing. The GO pathways of the differentially expressed mRNAs were mainly enriched in the ‘hormone metabolic process’, ‘cell wall organization’, ‘oxidoreductase activity’ and ‘catalytic activity’ categories. According to the gene expression analysis, significantly differentially expressed genes included Solyc02g080530.3 (Peroxide, POD), Solyc01g008710.3 (Mannan endo-1,4-beta-mannosidase, MAN), Solyc08g077910.3 (Expanded, EXP), Solyc09g075330.3 (Pectinesterase, PE), Solyc07g055990.3 (Xyloglucan endotransglucosylase-hydrolase 7, XTH7), Solyc12g011030.2 (Xyloglucan endotransglucosylase-hydrolase 9, XTH9), Solyc10g080210.2 (Polygalacturonase-2, PG2), Solyc08g081010.2 (Gamma-glutamylcysteine synthetase, gamma-GCS), Solyc09g008720.2 (Ethylene receptor, ER), Solyc11g042560.2 (Ethylene-responsive transcription factor 4, ERF4) etc. In addition, the lncRNAs (XLOC_16662 and XLOC_033910, etc) regulated the expression of their neighbouring genes, and genes related to tomato cracking were selected to construct a lncRNA-mRNA network influencing tomato cracking.Conclusions: This study provides insight into the responsive network for water-induced cracking in tomato fruit. Specifically, lncRNAs regulate the hormone-redox-cell wall network, including plant hormone (auxin, ethylene) and ROS (H₂O₂) signal transduction and many cell wall-related mRNAs (EXP, PG, XTH), as well as some lncRNAs (XLOC_16662 and XLOC_033910, etc.).

Soluble sugar is known to improve the sweetness and increase tomato sauce yield. Studies have focused on improving the content of soluble sugar in tomato fruits, usually by promoting functional genes. We studied two genes (SlINVINH1 and SlVPE5) that inhibited the accumulation of soluble sugar in tomato fruits and obtained two genes’ knocked-out lines (CRISPR-invinh1 or CRISPR-vpe5) using CRISPR/Cas9. Aggregated lines with CRISPR-invinh1 and CRISPR-vpe5 were gained by hybridization and self-pollination. Compared to wild-type lines, the glucose, fructose, and total soluble solid (TSS) contents of CRISPR-invinh1 and CRISPR-vpe5 increased significantly. Glucose, fructose, and TSS levels further improved simultaneously with CRISPR-invinh1 and CRISPR-vpe5 than with single gene knock-out lines. This indicates that these genes have a synergistic effect and will increase the soluble sugar content. Thus, the knock-out SlINVINH1 and SlVPE5 may provide a practical basis for improving the sweetness of tomato fruits and their processing quality.

Background: Late embryogenesis abundant (LEA) proteins are widely distributed in higher plants and play crucial roles in regulating plant growth and development processes and resisting abiotic stress. Cultivated tomato (Solanum lycopersicum) is an important vegetable crop worldwide; however, its growth, development, yield, and quality are currently severely constrained by abiotic stressors. In contrast, wild tomato species are more tolerant to abiotic stress and can grow normally in extreme environments. The main objective of this study was to identify, characterize, and perform gene expression analysis of LEA protein families from cultivated and wild tomato species to mine candidate genes and determine their potential role in abiotic stress tolerance in tomatoes. Results: Total 60, 69, 65, and 60 LEA genes were identified in S. lycopersicum, Solanum pimpinellifolium, Solanum pennellii, and Solanum lycopersicoides, respectively. Characterization results showed that these genes could be divided into eight clusters, with the LEA_2 cluster having the most members. Most LEA genes had few introns and were non-randomly distributed on chromosomes; the promoter regions contained numerous cis-acting regulatory elements related to abiotic stress tolerance and phytohormone responses. Evolutionary analysis showed that LEA genes were highly conserved and that the segmental duplication event played an important role in evolution of the LEA gene family. Transcription and expression pattern analyses revealed different regulatory patterns of LEA genes between cultivated and wild tomato species under normal conditions. Certain S. lycopersicum LEA (SlLEA) genes showed similar expression patterns and played specific roles under different abiotic stress and phytohormone treatments. Gene ontology and protein interaction analyses showed that most LEA genes acted in response to abiotic stimuli and water deficit. Five SlLEA proteins were found to interact with 11 S. lycopersicum WRKY proteins involved in development or resistance to stress. Virus-induced gene silencing of SlLEA6 affected the antioxidant and reactive oxygen species defense systems, increased the degree of cellular damage, and reduced drought resistance in S. lycopersicum. Conclusion: These findings provide comprehensive information on LEA proteins in cultivated and wild tomato species and their possible functions under different abiotic and phytohormone stresses. The study systematically broadens our current understanding of LEA proteins and candidate genes and provides a theoretical basis for future functional studies aimed at improving stress resistance in tomato.

Background: Sugar and organic acids not only contribute to the formation of soluble solids (Brix) but also are an essential factor affecting the overall flavor intensity. However, the possible metabolic targets and molecular synthesis mechanisms remain to be further clarified.Methods: UHPLC-HRMS (ultrahigh-performance liquid chromatography and high-resolution mass spectrometry) combined with comparative transcriptome analysis were performed in fruits at green ripe (S1), turning-color (S2), and red ripe (S3) stages of two tomato genotypes TM-1 (Solanum galapagense L., LA0436) and TM-38 (S. lycopersicum L. cultivar M82, LA3475) that vary in fruit Brix.Results: The fruit Brix of TM-1 was nearly twice that of TM-38 at S3. Nevertheless, TM-1 accumulated 1.84- and 2.77-fold the L-malic acid and citric acid in red ripe fruit (S3) compared with TM-38, respectively. D-glucose and D-fructose in TM-1 and TM-38 fruits tended to be similar at S3. Concomitantly, the sugar/organic acid ratio of TM-38 fruits were 23. 08-, 4. 38-, and 2.59-fold higher than that of TM-1 fruits at S1, S2, and S3, respectively. Among starch and sucrose (carbohydrate, CHO) metabolism (ko00500) genes, SUS (Solyc07g042550.3) and BAM (Solyc08g077530.3) were positively (r = 0.885–0.931) correlated with the sugar/organic acid ratio. Besides, INV (Solyc09g010080.3 and Solyc09g010090.5.1), AAM (Solyc04g082090.3), 4-α-GTase (Solyc02g020980.2.1), BGL2 (Solyc06g073750.4, Solyc06g073760.3, and Solyc01g081170.3), TPS (Solyc01g005210.2 and Solyc07g006500.3), and TPP (Solyc08g079060.4) were negatively (r = −0.823 to −0.918) correlated with the sugar/organic acid ratio. The organic acid (TCA cycle) metabolism (ko00020) gene ALMT (Solyc01g096140.3) was also negatively (r = −0.905) correlated with the sugar/organic acid ratio.Conclusion: Citric acid may play a more dominant role in the sugar/organic acid ratio of the tomato fruit, and the contribution of both L-malic acid and citric acid to the fruit Brix was much greater than that of D-glucose and D-fructose. Genes involved in CHO and TCA metabolism, which have a significant correlation with the sugar/organic acid ratio were considered to be the contributing factors of fruit Brix.