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Journal: Plants

Article Title: Genome Editing of the NF-YA8 Gene Modifies Tomato Plant Architecture and Fruit Traits

doi: 10.3390/plants14121826

Figure Lengend Snippet: Mutation screening of the target gene NF-YA8 . ( A ) DNA from chimeric founder plants (M1 generation; C1–C5) was screened for mutations using PCR amplification followed by agarose gel electrophoresis. Wild-type DNA (WT) and a 100 bp ladder (L) served as controls. NC: negative control. High-resolution melting (HRM) analysis further identified NF-YA8 mutations, as evidenced by shifted DNA melting curves in M1 plants relative to the wild-type control (red curve; lower panel). ( B ) The same PCR/electrophoresis and HRM analysis was performed on DNA from M2 plants ( nf-ya8-V , -T , -S , -Y , -A). Again, WT DNA and a 100 bp ladder (L) were used as controls, and the lower panel shows shifted melting curves in M2 plants compared to the wild-type control (red curve). ( C ) A multiple sequence alignment compared the NF-YA8 variants identified in the M1 and M2 generations, resulting from ZFN-induced mutations. The ZFN target sites are indicated by boxes. The following abbreviations denote mutations: "s" for substitution, "d" for deletion, and "ins" for insertion. Insertions are highlighted in blue, deletions are represented by red dashes, and substitutions are shown with red letters.

Article Snippet: DNA sequence alignments were visualized using Benchling ( www.benchling.com ).

Techniques: Mutagenesis, Amplification, Agarose Gel Electrophoresis, Negative Control, Control, Electrophoresis, Sequencing

Journal: Plants

Article Title: Genome Editing of the NF-YA8 Gene Modifies Tomato Plant Architecture and Fruit Traits

doi: 10.3390/plants14121826

Figure Lengend Snippet: Sequences of selected ZFN-induced mutations in M1 and M2 plants. ( A ) The C2 and C3 samples exhibited a biallelic mutation at position 524 (T to C) in exon 5 situated 165 nucleotides upstream of the binding site of the coding sequence, resulting in an amino acid substitution, 175L to P. Additionally, a C deletion at position 581 in exon 5 was identified in C2 RC. ( B ) In exon 6, a C-to-T transition at position 692 was observed, leading to an amino acid change, 231A to V. Notably, the 692C-to-T mutation occurred within the ZFN target site (689–713). ( C ) The C2 sample exhibited a deletion of adenine at position 769 in exon 6, which is 56 nucleotides downstream of the ZFN site, leading to the amino acid modification 256R. These sequence modifications, occurring within and near the genome editing target region, may have functional implications for NF-YA8 gene regulation and tomato development. Symbols and abbreviations: RC, reverse complement; S, sample; P, primer. ( D ) Chromatograms showing the detected mutations. ( E ) Overview of the locations of the mutations within the protein’s conserved domains, including the well-conserved core domain with predicted A1 and A2 helices, the NF-YB/NF-YC interaction domain, the DNA binding region, and the ZFN target site. A SWISS-MODEL alignment of the NF-YA8 amino acid sequence (Model_01) with its template shows the predicted A1 and A2 helices enclosed in rectangles. Τhe predicted structure from AlphaFold was used to compare the original (WT) and mutated forms from M1 (M1-C3) and M2 (Vigorous) generations. The analysis focused on structural differences near the A1 and A2 helices. The native structure features L175 and A231, while the mutant clone C3 contains P175 and the Vigorous A231. Insets (squares) provide close-up views highlighting these residue-specific changes (circles).

Article Snippet: DNA sequence alignments were visualized using Benchling ( www.benchling.com ).

Techniques: Mutagenesis, Binding Assay, Sequencing, Modification, Functional Assay, Residue