Journal: BMC Plant Biology

Article Title: Medicago Mting1 Mting2 double knockout mutants are extremely dwarfed and never flower implicating essential MtING functions in growth and flowering

doi: 10.1186/s12870-025-06432-x

Figure Lengend Snippet: Description of Mting1 and Mting2 single and double mutants

Article Snippet: Previously, we showed that Mting2 single knockout mutations negatively impacted growth and delayed flowering in Medicago , but Mting1 single mutants were like WT [ ].

Techniques: Mutagenesis, Knock-Out, Double Knockout

Journal: BMC Plant Biology

Article Title: Medicago Mting1 Mting2 double knockout mutants are extremely dwarfed and never flower implicating essential MtING functions in growth and flowering

doi: 10.1186/s12870-025-06432-x

Figure Lengend Snippet: Mting1-7 Mting2-2 double knockout mutants are very small and never flower. A Flowering time in days to flowering and ( B ) nodes to flowering in WT, Mting1-7 , Mting2-2 , and Mting1-7 Mting2-2 in VLD. C-H Graphs showing six morphological traits; the distance from the monofoliate to furthest trifoliate (plant spread; C ), total number of compound leaves ( D ), width ( E ), and height ( F ) of the largest terminal leaflet, primary axis height ( G ), and percentage of atypical (non-trifoliate) compound leaves on the plant ( H ) after 21 days in VLD. Data is shown as a boxplot with median and interquartile range. Statistical significance was determined using a Wilcoxon test ( A , B , H, I, J ; p -value with Bonferroni correction: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001) or one-way ANOVA and Tukey honest significant test ( C-G ). Groups with different letters are statistically different from each other. I The total chlorophyll concentration in µg per g of fresh weight and ratio of chlorophyll a to b ( J ) in the compound leaves of 33 day old WT, Mting1-7 and Mting2-2 or 233 day old Mting1-7 Mting2-2 grown in VLD. Data is mean (red cross) and 95% confidence interval (whiskers) with replicates shown as dots. Statistical difference from WT was tested using a t-test assuming unequal variance. K Photographs from above of WT, Mting1-7 , Mting2-2 , and Mting1-7 Mting2-2 after 14 days in VLD or from the side after 33 days in VLD ( L ). M The Mting1-7 Mting2-2 mutant after 75 days and 125 days in VLD. N Comparison between trichomes on a terminal leaflet adaxial surface in WT and Mting1-7 Mting2-2 . Scale bars are 1 cm unless labelled otherwise

Article Snippet: Previously, we showed that Mting2 single knockout mutations negatively impacted growth and delayed flowering in Medicago , but Mting1 single mutants were like WT [ ].

Techniques: Double Knockout, Concentration Assay, Mutagenesis, Comparison

Journal: BMC Plant Biology

Article Title: Medicago Mting1 Mting2 double knockout mutants are extremely dwarfed and never flower implicating essential MtING functions in growth and flowering

doi: 10.1186/s12870-025-06432-x

Figure Lengend Snippet: Mting1-1 Mting2-11 double PHD mutants are smaller and later flowering than WT. A Flowering time in days to flowering and ( B ) nodes to flowering in WT, Mting1-1 , Mting2-11 , and Mting1-1 Mting2-11 in VLD. C Graphs showing the total number of compound leaves and ( D ) percentage of atypical (non-trifoliate) compound leaves on the plant after 21 days in VLD. E Graph showing the height of the primary axis over time in VLD. Data is shown as a boxplot with median and interquartile range. Statistical significance was determined using a one-way ANOVA and Tukey honest significance test ( A-C ) (Groups with different letters are significantly different) or Wilcoxon test ( D , E ; p -value with Bonferroni correction: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). F Photograph showing WT, Mting1-1 , Mting2-11 and Mting1-1 Mting2-11 plants. G Typical compound leaf and ( H ) the adaxial leaf surface comparing trichomes on WT and Mting1-1 Mting2-11 after 32 days in VLD. Scale bars are 1 cm unless labelled otherwise

Article Snippet: Previously, we showed that Mting2 single knockout mutations negatively impacted growth and delayed flowering in Medicago , but Mting1 single mutants were like WT [ ].

Techniques:

Journal: BMC Plant Biology

Article Title: Medicago Mting1 Mting2 double knockout mutants are extremely dwarfed and never flower implicating essential MtING functions in growth and flowering

doi: 10.1186/s12870-025-06432-x

Figure Lengend Snippet: MtING proteins are nuclear localized but do not interact in vitro to form dimers. A Schematic diagram showing the location of the nuclear localization signal (NLS) domain in HsING4. B Protein sequence alignment of the human HsING4 NLS domain and the homologous Medicago ING regions and ( C ) end of the PHD finger. The HsING4 NLS (black line) and nucleolar localization sequence (NoLS, grey lines) identified by Tsai et al. are shown. The numbering is based on the relative position within ING4. D A table showing potential predicted NLS peptides using NLStradamus . E Confocal microscopy images from tobacco explants co-transformed with NLS:mCherry (red channel, magenta) and either GFP , MtING1:GFP or MtING2:GFP (green channel, green) overexpression constructs. Photographs are representative of two separate experiments each with three independent transformations. Scale is 100 µM. F SEC–MALLS was used to analyse if tagged MtING proteins dimerised in vitro. Graph showing the elution profile for TRX-tagged MtING1 ING domain (TRX:MtING1 ING ) and ( G ) MBP-tagged MtING2 ING domain (MBP:MtING2 ING ) recombinant protein at a concentration of ~ 4.25 mg/ml (289 µM or 62.8 µM for ING1 or ING2, respectively) or ( H ) 1:1 equimolar (51.4 µM) mixture of MBP:ING2 ING and TRX:MtING1 ING . The protein elution profile (light blue) is measured by the change in refractive index (∆RI) and the calculated weight-average molecular weight (dark blue) is in kilodaltons. The expected molecular weight of monomeric tagged MtING ING protein is indicated by a dashed grey line

Article Snippet: Previously, we showed that Mting2 single knockout mutations negatively impacted growth and delayed flowering in Medicago , but Mting1 single mutants were like WT [ ].

Techniques: In Vitro, Sequencing, Confocal Microscopy, Transformation Assay, Over Expression, Construct, Recombinant, Concentration Assay, Refractive Index, Molecular Weight

Journal: BMC Plant Biology

Article Title: Medicago Mting1 Mting2 double knockout mutants are extremely dwarfed and never flower implicating essential MtING functions in growth and flowering

doi: 10.1186/s12870-025-06432-x

Figure Lengend Snippet: Gene expression in Mting1-7 Mting2-2 is consistent with non-flowering and has similarities to Atepl1 . A PCA plots for apex and leaf tissue RNA-seq data from the single parental Mting mutant lines, double Mting mutants and WT. Three biological replicates were harvested for each genotype. B A heat map of the log2 fold change obtain with the deseq2 analysis of all up and down differentially expressed genes in Mting1-7 , Mting2-2 and Mting1-7 Mting2-2 relative to WT and ( C ) 93 selected candidate genes extracted by Cheng et al . in all mutants relative to WT. The data is represented as the log2 of Mutant TPM /WT TPM . D Boxplot of the log2( Mutant TPM /WT TPM ) in all Mting mutants for Medicago homologs of Arabidopsis genes targeted by NuA4. The Arabidopsis genes were previously identified as targets of NuA4 by RNA-seq and ChIP-seq in the Atepl1 NuA4 mutant and WT . E RT-qPCR analysis (left panel) and RNA-seq (right panel) of 21 candidate flowering genes in Mting mutant apex tissue. The left panel shows the boxplot of log2 fold-change relative to WT. Asterisks indicate a significant difference in gene expression between WT and mutant by t-test, assuming unequal variance ( P ≤ 0.05). The right panel is expression shown as Z scores extracted from the TPM values of the RNA-seq

Article Snippet: Previously, we showed that Mting2 single knockout mutations negatively impacted growth and delayed flowering in Medicago , but Mting1 single mutants were like WT [ ].

Techniques: Gene Expression, RNA Sequencing, Mutagenesis, ChIP-sequencing, Quantitative RT-PCR, Expressing