Combined transcriptome and plant hormone analysis revealed the potential mechanism of fern-leaved tomato mutant formation

To investigate the molecular regulation mechanisms and metabolic pathways associated with tomato leaf abnormalities, we utilized the leaves of wild-type Alisa Craig (AC) and the fern-leaved tomato mutant (fern mutant) as experimental materials. Phenotypic identification revealed that the young leaves of the fern mutant presented strip-shaped ((Fern), similar to fern leaves., while the mature leaves reverted to a morphology similar to that of AC leaves (referred to as Fern-NL). However, the leaf cleavage in Fern-NL was not pronounced, and the fern mutant exhibited dwarfism with shortened internode lengths. Microscopic analysis of the leaves from both Fern and Fern-NL, using AC as a control, indicated that the upper epidermal cells of Fern were elongated, the lower epidermal cells were irregularly arranged, the palisade tissue was densely packed, and the spongy tissue was sparse. In contrast, no significant differences were observed between the upper and lower epidermis of Fern-NL and that of AC; both exhibited tightly arranged palisade tissue and densely but irregularly arranged spongy tissue. Transcriptome sequencing data analysis using DEGsq revealed 2525 (157 up-regulated, 955 down-regulated) genes differentially expressed in AC vs Fern, 4864 (1679 up-regulated, 3185 down-regulated) in Fern vs Fern-NL, and 5341 (2151 up-regulated, 319 down-regulated) in AC vs Fern-NL. Functional analysis indicated significant differences in DEGs related to intrinsic components of the plasma membrane, ion transmembrane transport activity, and stimulation response. KEGG enrichment analysis highlighted active enrichment of DEGs in pathways such as plant hormone signal transduction, phenylpropanoid biosynthesis, plant-pathogen interaction, and carbon metabolism, suggesting their potential role in fern-leaved tomato mutant formation. Quantitative hormone analysis revealed significant alterations in auxin, cytokinin, gibberellin, abscisic acid, ethylene synthesis precursors, jasmonic acid, salicylic acid, and zeatin levels in the Fern mutant. Treatment with auxin promoter NAA and inhibitor TIBA demonstrated Fern's heightened sensitivity to NAA and TIBA, with NAA promoting leaf recovery and TIBA delaying it. Comprehensive analysis indicates that differentially expressed genes regulating the auxin signaling pathway act in concert with imbalances in hormones such as jasmonic acid and abscisic acid, leading to abnormal arrangement of epidermal cells and densification of palisade tissue, which ultimately drives the formation of fern-like leaf morphology. These findings offer insights into the molecular mechanisms underlying tomato leaf dysplasia and serve as a valuable resource for germplasm innovation, genetic resource exploration, and cultivation strategies.