## 2 Results
### Differentially Expressed Genes Among Samples and Groups  
The fruit from the fourth generation of mutant lines M7_5 and M29_2 was chosen for RNA sequencing, while differential gene expression analysis was performed against the WT fruit (Heinz 1706). Principal component analysis (PCA) confirmed the inter-group variation and intra-group consistency, supporting the selection of these mutant lines for comparative analysis (Figure 2A). Gene expression analysis revealed distinct patterns across genotypes using hierarchical clustering. The heatmap (Figure 2B) displays differential gene expression, with the mutant lines (M7_5 and M29_2) forming separate clusters from the WT control. Notably, M7_5 exhibited a largely reversed expression pro  
Figure 1: Morphometric traits of tomato fruits. (**A**) Impact of L1L4 disruption on fruit composition of the mutant lines M7_5 and M29_2 (based on [9]). TA: titratable acidity. Higher is shown by upward arrows, lower by downward arrows. (**B**) Morphometric variation in fruits from the wild-type (WT) line, and the two disruption lines M7_5 and M29_2 for distal end protrusion, distal angle micro, shape (ovoid), and pericarp thickness. Data are presented as means \(\pm\) SD, \(n\) = 13–19, with the statistical significance determined by a one-way ANOVA. The differences among the groups were determined using [PERSON]’s post hoc test for multiple comparisons. The significance levels are indicated by asterisks as follows: \({}^{**}p<0.001\), \({}^{***}p<0.0001\), and \({}^{***}p<0.0001\).  
file compared to the WT, with genes highly expressed in the WT showing significantly reduced expression in M7_5, and vice versa. Volcano plots visualized the distribution of DEGs between mutants and the WT (Figure 2C,D). A Venn diagram (Figure 2E) highlighted the overlap (5021 genes) and unique gene expression profiles for each mutant group (M7_5: 9522 genes; M29_2: 6850 genes) relative to the WT. To identify the genes regulated by the ZFN-induced gene disruption, differential expression analysis was performed using DESeq2 (R package 1.20.0). Significant DEGs were defined as those with an FDR < 1 \(\times\) 10\({}^{-3}\), 1 log2 FoldChange \(\mid\)\(\geq\) 1, and an adjusted _p_-value \(\leq\) 0.05, while excluding the genes with low expression levels (<50). Mapping short reads to the tomato reference genome allowed for the quantification of DEGs in each mutant compared to the WT, and between the mutant lines. Pairwise comparisons revealed the following: M7_5 vs. WT showed 9522 DEGs (2171 upregulated, 5661 downregulated) (Figure 2C); M29_2 vs. WT showed 6852 DEGs (3000 upregulated, 3850 downregulated) (Figure 2D); and M7_5 vs. M29_2 showed 5892 DEGs (2171 upregulated, 3721 downregulated). The Venn diagram (Figure 2E) illustrates the shared and unique differential gene expression (DEGs) between M7_5 vs. WT and M29_2 vs. WT (5021 shared DEGs). The greater number of DEGs in the M7_5 vs. WT comparison compared to M29_2 vs. WT suggests that the M7_5 mutation had a more pronounced impact on target gene expression. Conversely, the fewer DEGs in M29_2 vs. WT indicate a milder effect of the ZFN-targeted disruption in the M29_2 mutant.  
Our data highlight L1L4's critical role as a wide regulator of gene expression in tomato fruit, with a total of approximately 5000 shared DEGs between the mutant lines, and at least 3000 downregulated DEGs (Figure 2F), indicating a wider suppression than activation. In addition, both lines share 3084 downregulated DEGs and 1640 upregulated ones, indicating a particularly more suppressive role of L1L4 in the regulation of tomato fruit complex biological processes. On the other hand, M7_5's higher number of both total and unique DEGs reveals that the induced L1L4 mutations in line M7_5 have a more prominent transcriptional impact, compared to line M29_2.