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

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Despite this, the molecular mechanisms regulating flowering in the economically important Fabaceae (legume) family are not fully understood. For example, the key flowering regulators known from Arabidopsis, FLC and CO, do not regulate flowering in the temperate model legume Medicago truncatula (Medicago). Previously, we used CRISPR-Cas9 mutagenesis to show the histone modification reader MtINHIBITOR OF GROWTH 2 promotes flowering and growth in Medicago. However, surprisingly, the highly conserved C-terminal plant homeodomain (PHD) finger did not appear to contribute to this, as Mting2 PHD finger mutants flowered and grew similarly to wild type. Additionally, a second ING gene, MtING1 , did not appear to regulate flowering. Methods To further dissect the genetic function of the two MtING genes in flowering and growth, we cross-pollinated selected Mting1 and Mting2 single mutants to create two different double mutants; the Mting1-7 Mting2-2 double knockout mutant and the Mting1-1 Mting2-11 double PHD finger mutant. The growth and flowering of these mutants was assessed in floral-inductive vernalised long day conditions. We also used fluorescence confocal microscopy and in vitro protein biophysical analysis to investigate the subcellular localization and oligomerization of the proteins. Finally, we carried out gene expression analysis by RNA-seq and RT-qPCR to determine how the two genes affect transcript accumulation to influence growth and flowering. Results The Mting1-7 Mting2-2 double knockout mutants were very small, did not maintain outgrowing branches and never flowered. Mting1-1 Mting2-11 double PHD finger mutants on the other hand showed only mild dwarfing and delays to flowering. GFP tagged MtING proteins localised to the nucleus in tobacco leaves. However, recombinant MtING domain proteins did not form dimers in solution. Gene expression analyses showed large changes to global gene expression in the double knockout mutant with key flowering genes downregulated and predicted floral repressors elevated. Conclusions Although the first ING gene was identified in humans almost three decades ago, very little is known about the two plant ING genes. Our findings demonstrate the essential combined role the MtING genes play in the regulation of gene expression, flowering time and wider development. INHIBITOR OF GROWTH MtING1 MtING2 legume Medicago Arabidopsis flowering time PHD finger ING domain Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The timing of flowering is a major factor in successful plant adaptation and crop productivity, including in the legume family which is the second most economically important plant group after the cereals [ 1 ]. Like winter annual Arabidopsis, the temperate model legume Medicago truncatula (Medicago) is induced to flower by extended cold (vernalization, V) followed by warm long day (LD) conditions (VLD) [ 2 ]. However, strikingly in Medicago there is no CONSTANS that promotes flowering in LD, or FLOWERING LOCUS C to repress flowering like in Arabidopsis [ 3 – 6 ]. In addition, a predicted Polycomb complex PRC2 component MtVRN2 has a different function in flowering in Medicago to Arabidopsis, repressing the important FLOWERING LOCUS T (FT) -like gene MtFTa1 prior to vernalization [ 7 ]. Despite this, other Medicago homologs appear to promote or repress flowering in a similar manner to Arabidopsis, such as the Medicago floral activators MtFTa1 , SUPPRESSOR OF CONSTANS 1 ( SOC1 ) MtSOC1a , the photoreceptor MtPHYTOCHROME A which strongly promotes flowering in LD and MtFE , or repressors such as MtCYCLING DOF FACTORS [ 8 – 12 ]. Furthermore, the FT-FLOWERING LOCUS D (FD) complex is crucial for flowering in Medicago as double Mtfta1 Mtfda mutants never flower [ 13 ]. Gene edited Mtsoc1a Mtsoc1b Mtsoc1c triple mutants also do not transition to flowering, implicating a broader role for the duplicated MtSOC1 -like genes in Medicago than SOC1 in Arabidopsis [ 9 , 14 ]. INHIBITOR OF GROWTH ( ING ) genes are found in most eukaryotes [ 15 ]. First identified as a tumour suppressor in humans [ 16 ], they have since been studied in other organisms such as yeast and are known to be involved in the regulation of gene transcription and development [ 17 , 18 ]. The genes encode proteins with two conserved domains, a N-terminal ING domain and a C-terminal plant homeodomain (PHD) finger. The ING domain has been shown to facilitate protein-protein interactions, including to recruit activator histone acetyltransferase or repressor histone deacetylase complexes to genes influencing their transcription [ 19 – 21 ]. For example, in yeast the YNG2 ING protein is a part of the NuA4 histone acetyltransferase [ 21 – 23 ]. The C-terminal PHD domain is a Cys4-His Cys3 zinc finger that binds to the chromatin mark H3K4me3 [ 24 ]. Many plants, including Medicago and Arabidopsis, have two ING genes; ING1 and ING2 [ 25 – 27 ]. They encode proteins which are highly similar between plant species, with the Arabidopsis and Medicago proteins sharing 68% (ING1) to 80% (ING2) sequence identity, while only being ~ 30% identical between ING1 and ING2 within species [ 27 ]. The Arabidopsis ING proteins are present in the nucleus and can bind H3K4me3 peptides in vitro [ 25 , 26 ]. In addition, binding studies and mass spectrometry indicate that Arabidopsis ING2 may be a part of a NuA4 complex, functioning as a scaffold in the Piccolo catalytic module [ 28 – 31 ]. No Arabidopsis Ating mutant studies have been published yet, but recently we showed in Medicago that gene-edited Mting2 single knockout mutant plants were pale in colour and dwarfed [ 27 ]. They also had reduced MtFTa1 expression and delayed flowering, especially in floral inductive VLD conditions compared to wild type (WT) plants [ 27 ]. Analysis of a range of different gene-edited Mting2 mutant plants indicated that an intact MtING2 ING domain was important for WT-like growth, development and flowering. However, unexpectedly, we saw that mutations that affected the C-terminal PHD finger of MtING2, but left the ING domain intact, had little effect on plant growth and development. Gene edited Mting1 single knockout mutants also grew and flowered similar to WT [ 27 ]. Here we make and characterise the Mting1-7 Mting2-2 double knockout mutant which has striking abnormal growth phenotypes and never flowers, indicating genetic redundancy as well as complementary functions. We identify differentially expressed genes by RNA-seq and RT-qPCR and compare these to Arabidopsis genes identified as potential NuA4 histone acetyltransferase targets [ 31 ]. We also investigate possible genetic redundancy of the PHD finger encoded by the ING genes by evaluating the Mting1-1 Mting2-11 double PHD mutant. Methods Plant material and growth. Wild type Medicago R108_C3 (WT) was used in this study [ 32 ]. The Mting1 and Mting2 single mutants: Mting1-1, Mting1-7, Mting2-2 and Mting2-11 were previously reported by Jaudal et al. [ 27 ] (Table 1 ). Mting1 Mting2 double mutants were generated by crossing in this study (Table 1 ). To generate Mting1-7 Mting2-2 double knockout mutant and the Mting1-1 Mting2-11 double PHD finger mutant, the respective Mting1 and Mting2 mutant plants were manually cross-pollinated as previously described [ 33 ]. The combination of Mting alleles in the subsequent segregating F1 and F2 progeny was confirmed using allele specific genomic PCR (Table S1 ). Table 1 Description of Mting1 and Mting2 single and double mutants line predicted mutation effect mutation description predicted length (aa) phenotypes Mting1-7 predicted knockout deletion causing a frame shift, encoding highly truncated protein 83 WT-like* Mting2-2 predicted knockout large deletion causing a frame shift, encoding highly truncated protein 44 small, late flowering, pale* Mting1-1 PHD finger deletion large deletion, removing encoded PHD finger 172 WT-like* Mting2-11 PHD finger deletion small indels, affecting encoded ING domain loop region and removing PHD finger 195 WT-like* Mting1-7 Mting2-2 double knockout mutant as above tiny, no branches, never flowers Mting1-1 Mting2-11 double PHD finger mutant as above mild developmental and flowering phenotypes The predicted length of WT MtING1 is 247 amino acids (aa), and MtING2 is 263 aa. *according to Jaudal et al . [ 27 ] All Medicago plants used for this research were grown under VLD which is a floral-inductive condition. To sow plants, seeds were scarified using sandpaper (p600 grit), sterilised in a chlorine solution (Millipore, USA) for 10 minutes and germinated overnight, shaking in water at 15°C in the dark. Seedlings were vernalized for 3 weeks at 4°C on moist filter paper in a petri dish. Vernalized seedlings were planted directly into seed-raising mix (Daltons, NZ) in small 6-cell punnets placed on rockwool mats (Grodan, NE) sub irrigated with hydroponics [ 34 ] (without Na 2 O 3 Si). The plants were grown in a controlled greenhouse under white fluorescent light (~ 160–200 µmol m − 2 s − 1 ) in long days (16/8 hours light/dark) at 22°C. Nicotiana tabacum (tobacco) was used for transient expression of 35S:MtING-GFP , 35S-eGFP and 35S:NLS-mCherry . Seeds were sown at high density directly into seed raising mix and healthy seedlings were transplanted into 0.5 L pots with planting mix (Daltons) watered with hydroponics solution. The tobacco plants were grown under the same conditions as the Medicago. Phenotypic analysis. The flowering time of plants was measured by the number of days after planting, and the number of nodes on the primary axis at the emergence of the first floral bud on the plant. For the flowering time data presented in Fig. 1 , the sample size (n) ranged from 4 to 24 and in Fig. 2 , 8 to 16. All other data is presented at a stated plant age where n = 6 to 11 for the data in Fig. 1 , or 8 to 16 for Fig. 2 . The plant spread was measured as the horizontal distance in millimetres from the monofoliate leaf tip to the leaf tip of the furthest branch. The total number of leaves was measured as the total number of fully expanded compound leaves plus the monofoliate leaf on the plant. The length of the primary axis was measured as the distance in millimetres from the base of the monofoliate node to the growth tip. The percentage of atypical leaves was measured as the number of non-trifoliate compound leaves (excluding the monofoliate) on the plant divided by the total leaf number. All phenotypic data is graphed as a boxplot with median and interquartile range. Statistical significance was tested with a one-way ANOVA and Tukey honest significance test (p-value ≤ 0.05) or where the data does not satisfy the assumptions of an ANOVA, a Wilcoxon test with Bonferroni correction testing for a difference to WT. Chlorophyll measurements. Chlorophyll content was measured according to Porra et al . [ 35 ] with modification. Chlorophyll was extracted from two leaves from a plant for each of the three biological replicates after 33 days in VLD for WT, Mting1-7 and Mting2-2 , or three replicates from different leaves on one Mting1-7 Mting2-2 double mutant plant after 233 days. Two of the newest fully expanded compound leaves on the primary axis were harvested, weighed (approximately 100–150 mg) and frozen in liquid nitrogen. Chlorophyll was extracted from ground tissue using 2–5 ml of Tris-HCl pH 7 buffered 80% acetone. Samples were processed four at a time and diluted in additional buffered acetone so the absorbance at 663 nm was between 0.5 to 0.8. The absorbance at 646 nm and 663 nm (with correction at 750 nm) was measured using a Cary 4000 spectrophotometer (Agilent, USA). The total chlorophyll per gram of fresh weight and the chlorophyll a to b ratio was calculated according to Porra et al . [ 35 ]. The statistical significance from WT was tested using a t-test assuming unequal variance. Fluorescent protein localization assay. The full-length MtING1 or MtING2 coding sequence was cloned into a pHEX2 vector [ 36 ] modified for fusion with eGFP at the C-terminal end of the encoded proteins under the cauliflower mosaic virus constitutive 35S promoter. A control 35S:eGFP was also cloned into pHEX2. A positive nuclear targeted control 35S:NLS:mCherry was also cloned using Golden Gate assembly [ 37 ]. The vectors were transformed independently into Agrobacterium tumefaciens strain GV3101 and cultured. A. tumefaciens containing the respective GFP and mCherry vector were mixed in equal parts and resuspended into infection medium (50 mM MES pH 5.6, 2 mM Na 2 HPO 4 , 28 mM glucose, 200 µM acetosyringone), and injected into the two newest leaves of 5–7 leaf stage tobacco plants. Small circular discs were sampled from the infiltrated leaves after three or five days. Transformations were repeated in two independent experiments with three independently transformed plants per experiment. Confocal laser scanning microscopy images were captured on an Eclipse Ti-E microscope with a 40x S Plan Fluor ELWD objective lens (Nikon, Japan), CSU-X1 confocal scanner unit (Yokogawa, Japan), ALC-500 laser unit and Zyla sCMOS camera (Andor, UK). GFP and mCherry were excited using a laser light wavelength of 488 nm and 561 nm, respectively, and images captured with a 3035B and TX Red 4040B filter (Semrock, USA). For each sample, greater than 100 cells from each leaf disc were observed. Single slice images were processed using NIS-Elements imaging software (Nikon, v4.06.12). Potential nuclear localization signal (NLS) sites for the MtING proteins were identified using NLStradamus [ 38 ]. The full-length sequences of human ING4, MtING1 and MtING2 were queried using a 4-state HHM static model with 0.6 threshold on the NLStradamus webserver [ 38 ]. SEC-MALLS. Size exclusion chromatography (SEC) with multi angle laser light scattering (SEC-MALLS) was used to determine the weight-average molecular weight (MW) and oligomeric state of recombinant MtING ING domain protein in solution. The coding sequence of the ING domain of MtING1 and MtING2, named MtING1 ING (M1-E118) and MtING2 ING (M1-E133), was cloned from WT into a pET-49b(+) vector for N-terminal fusion with either THIOREDOXIN (TRX:MtING1 ING ) or MALTOSE BINDING PROTEIN (MBP:MtING2 ING ). The induction of recombinant protein expression was the same as previously described [ 27 ]. Protein was purified from cell lysate using either nickel immobilised metal affinity chromatography (TRX:MtING1 ING ) or amylose affinity chromatography (MBP:MtING2 ING ) followed by SEC, and then concentrated to 4.25 mg/mL using an Amicon 3K centrifugal concentrator (Merck, De). Purified samples (100 µL) were analysed using SEC-MALLS at 25°C with a Dionex UltiMate 3000 HPLC pump (Thermo Fisher, USA), SLD7000 7-angle MALL detector (PSS, De), RI-101 differential refractive index detector (Shodex, De) and Superdex 200 Increase 10/300 GL 24 mL column (General Electric, USA) in MALLS buffer (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.1 mM TCEP, 3 mM azide). The MW was determined using PSS winGPC Unichrom software. Plant tissue harvesting and RNA extraction. For RNA-seq and real time reverse transcription quantitative PCR (RT-qPCR), leaf and shoot apex tissue was harvested from plants grown under VLD conditions, 4 h after dawn on day 14 (WT, Mting1-7 , Mting1-1 , Mting2-11 and Mting1-1 Mting2-11 ), day 15 ( Mting2-2 ) and day 21 ( Mting1-7 Mting2-2 ) when the plants had 2–3 fully expanded compound leaves. Three biological replicates were harvested per tissue type per genotype, each consisting of 1 to 2 fully expanded compound leaves from the same plant, or 3 to 4 primary apices from different plants. Harvested tissue was snap-frozen in liquid nitrogen and homogenised by metal beads in a Geno/Grinder® 2010 (New Jersey, USA). Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, De) according to the user manual. RNA quantity and quality were checked by a NanoPhotometer® N60 (Implen, De) and a Bioanalyzer 2100 (Agilent Technologies, USA). RNA-seq and analysis. The RNA-seq was carried out by Novogene (Hong Kong) with library preparation and sequencing as previously described [ 14 ]. Directional strand specific mRNA libraries (poly A enrichment) were prepared and sequenced on the Illumina NovaSeq platform NovaSeq6000, 150 bp paired-end (Novogene, Hong Kong). The FASTQ file read quality was evaluated, and Fastp (v0.21) was used for trimming [ 39 ]. Reads with a quality below a PHRED score of 20 were trimmed from the 3’end, and reads < 36 bp in length were excluded. The remaining reads were mapped against the Mt4.0v2 transcriptome [ 40 , 41 ] using Salmon (v0.8.2). DESeq2 (v1.24.0) [ 42 ] was used for normalization and differential expression analyses. Differentially expressed transcripts were filtered using a cut-off adjusted p-value ≤ 0.05 and log2 fold-change ≥ 1 or ≤ − 1. To identify candidate direct target genes of NuA4, we used Blastx [ 43 ] against Arabidopsis NuA4 bound and regulated genes [ 31 ]. All the genes with an e-value < e − 100 were selected. Genes that were not expressed or only weakly expressed were removed, which gave a list of 289 transcripts (Table S5 ). Gene expression analysis by RT-qPCR. The WT and Mting1-7 Mting2- 2 RNA samples were treated with DNase (TURBO DNA- free ™ Kit, Invitrogen, USA) according to manufacturer's instructions. Template cDNA was synthesised with SuperScript™ IV Reverse Transcriptase (Invitrogen) as previously described [ 8 , 10 ]. Gene expression analysis was performed based on the comparative CT method [ 44 ], with modifications [ 14 , 45 ]. ΔCT values were obtained by normalising genes of interest to the reference gene, PROTEIN PHOSPHATASE 2A ( PP2A , Medtr6g084690). Relative gene expression was calculated using the formula 2 −ΔCT . Statistical significance between WT and Mting1-7 Mting2-2 was calculated using the t-test, assuming unequal variance (p-value ≤ 0.05). RT-qPCR primers are listed in Table S1 . Results Mting1-7 Mting2-2 double knockout mutants have severe growth and developmental defects and never flower Previously, we showed that Mting2 single knockout mutations strongly impacted growth and delayed flowering in Medicago, but Mting1 single mutants were similar to WT [ 27 ]. To assess the physiological consequences of strong mutations in both MtING genes, we generated a double knockout mutant by crossing Mting1 and Mting2 single knockout mutants. The Mting1-7 single knockout mutant carried a deletion causing a frameshift in the encoded protein. This led to an altered amino acid (aa) sequence from aa 21 onwards and a truncated protein of 83 aa, compared to 247 aa WT MtING1 (Table 1 ). The small, pale green and late flowering Mting2-2 single knockout mutant was predicted to encode a truncated protein with a deletion of aa 16 to 39 and a final protein of 44 aa, compared to WT MtING2 of 263 aa (Table 1 ). After crossing, we grew F1 plants and then identified homozygous Mting1-7 Mting2-2 double mutant F2 progeny by PCR genotyping (Table S1 ). The double mutants had multiple highly abnormal phenotypes, compared to the single mutant parents and WT (Fig. 1 ). As expected, in the parental lines, Mting1-7 flowered like WT, while Mting2-2 flowered late in floral inductive VLD conditions. Conversely, the Mting1-7 Mting2-2 double knockout mutant never produced a floral bud (Fig. 1 A/B). The double mutants continued to grow very slowly, but still had not flowered after six months after which they started to die. The double mutants were severely dwarfed even when compared to the small Mting2-2 mutant (Fig. 1 K/L). The Mting1-7 Mting2-2 plants also had significantly reduced plant diameter (plant spread), smaller leaflets and fewer compound leaves compared to Mting2-2 (Fig. 1 C-F). The double mutants could not maintain outgrowing branches, because they withered as the plants grew (Fig. 1 M). Similar to the Mting2-2 single mutant parent [ 27 ], they had an increase in the percentage of atypical compound leaves and no leaf trichomes (Fig. 1 H/N). Therefore, this double mutant analysis implicates both MtING genes as essential for normal development in Medicago, with redundant and complementary roles in the regulation of flowering, growth and plant architecture. Otherwise, Mting1-7 single mutants had a slight increase in plant size when compared to WT in five of the measured morphological traits (Fig. 1 C-G) at 21 days in VLD. This indicates that Mting1-7 in an otherwise WT background appears to have a weakly promotive effect on Medicago plant size, unlike Mting2-2 mutants which are dwarfed compared to WT. Previously, we reported that Mting2 mutants had a pale colour [ 27 ]. To determine if this was the result of changes in chlorophyll levels, we measured the chlorophyll content in the leaves of the mutants compared to WT. Mting2-2 mutants had a reduced total chlorophyll amount compared to WT and Mting1-7 , but there was no change in the ratio of chlorophyll a to chlorophyll b for any plants indicating that Mting2 affects the production of both chlorophylls equally (Fig. 1 I/J). In contrast, the double knockout mutant displayed similar chlorophyll levels to WT (Fig. 1 I/J). Mting1-1 Mting2-11 double PHD mutants show only mild growth and flowering phenotypes but a strong reduction in leaf trichomes Previously, our analysis of a range of gene-edited Mting2 single mutants indicated that the ING domain of MtING2 was important for normal growth and flowering in Medicago [ 27 ]. But, unexpectedly, Mting2 mutants with changes to or loss of the PHD finger domain grew similarly to WT, indicating that the MtING2 PHD finger domain was not necessary in an otherwise WT background [ 27 ]. As demonstrated above, Mting1-7 Mting2-2 double knockout mutants had a much stronger abnormal growth and development phenotypes than the parental lines, indicating that MtING1 and MtING2 had overlapping and complementary functions. Thus, we hypothesised that Mting double PHD finger mutants may also exhibit enhanced mutant phenotypes compared to the single PHD finger mutants. To test this, we crossed Mting2-11 plants with mutations predicted to only affect the PHD coding sequence and the non-essential loop region of the ING domain and which grew similarly to WT, to Mting1-1 plants with mutations predicted to remove large portions of the PHD finger, but not affecting the ING domain and which also grew like WT (Table 1 ) [ 27 ]. After crossing, we selected the Mting1-1 Mting2-11 double PHD finger mutants in the F2 generation by PCR genotyping (Table S1 ). Phenotyping of the Mting1-1 Mting2-11 double PHD mutants (Fig. 2 ) showed that they flowered slightly later in both days and nodes to flowering than WT and the parents (Fig. 2 A/B). The parental lines showed a mild delay to flowering in days, but not in nodes compared to WT. The Mting1-1 Mting2-11 double PHD mutants still flowered much earlier than the Mting2-2 knockout plants (Fig. 1 A/B). This suggested that both ING PHD fingers made a mild contribution to the promotion of flowering, but had a much weaker effect than the intact ING proteins. In addition, the Mting1-1 Mting2-11 double PHD mutant plants and their compound leaves appeared more compact than WT and the single mutant parental lines (Fig. 2 F/G), indicating again a mild contribution of both of the MtING PHD fingers on plant growth. Interestingly, the Mting1-1 Mting2-11 double PHD mutants also displayed greatly reduced visual leaf trichome density compared to WT (Fig. 2 H). The reduction in trichomes was similar to the Mting1-7 Mting2-2 double knockout mutants (Fig. 1 N). This implicates both MtING PHD fingers as vital for normal leaf trichome development. In other phenotypes, the Mting1-1 Mting2-11 double PHD mutant and the Mting2-11 parent had a similarly slightly shorter primary axis and a higher percentage of atypical compound leaves, compared to WT and Mting1-1 (Fig. 2 D/E). This suggested that loss of the MtING2 PHD finger alone was sufficient for this mild dwarfing and change in leaf patterning. Compared to WT, total leaf number was slightly reduced to a similar level in Mting1-1 and the Mting1-1 Mting2-11 double PHD mutant (Fig. 2 C). In summary, Mting1-1 Mting2-11 double PHD mutants had weakly abnormal growth and delayed flowering phenotypes and strong loss of leaf trichomes. MtING proteins localize to the nucleus in tobacco cells but do not dimerize in vitro Human ING proteins contain a nuclear localization signal (NLS) in the disordered linker between the PHD finger and N-terminal ING domain and are nuclear localized [ 46 ]. Furthermore, some human ING proteins contain an additional nucleolar localization signal (NoLS) within the NLS or downstream of the PHD finger [ 47 – 49 ]. Previously, it was shown that Arabidopsis AtING1 and AtING2 GFP-tagged proteins located to the nucleus in transient expression in protoplasts, although the NLS and NoLS identified in human INGs were not found in these ING proteins [ 25 , 26 ]. Here, we first compared the known NLS from human ING4 to the homologous regions of MtING1 and MtING2 and also used in silico analysis to predict NLS in the Medicago proteins (Fig. 3 A-D). Both of the Medicago INGs contain an enrichment of basic R and K residues in the NLS domain region which is similar to that of the human ING4 NLS (Fig. 3 B) [ 46 , 48 ]. Furthermore, MtING1, but not MtING2, has a RK rich sequence down downstream of the PHD finger, similar to the second NLS of ING4 (Fig. 3 C) [ 49 ]. This is supported by the in silico prediction which identified both sites as potential NLS for MtING1. No sites were predicted for MtING2 (Fig. 3 D). Therefore, to empirically test if MtING1 and MtING2 are nuclear localised, we generated overexpression constructs designed to express MtING1 and MtING2 translational fusions with C-terminal eGFP and used Agroinfiltration for transient expression in tobacco leaves. An eGFP construct was infiltrated as a positive GFP control, while a NLS-mCherry construct was co-transformed with the eGFP constructs and provided a positive nuclear control. Confocal microscopy (Fig. 3 E) indicated that both MtING1 and MtING2 fusion proteins are nuclear localized, like the control NLS-mCherry. In contrast the control eGFP was located to both nucleus and cytoplasm. Studies of three of the human ING proteins most closely related to the plant INGs have shown that they form homodimers and heterodimers through the N-terminal ING domain in solution [ 20 , 50 , 51 ]. Thus it is possible that plant ING proteins are also physically interacting through the ING domain. If the plant ING proteins do form homo or hetero dimers in planta this could help to explain why mutation of both MtING genes is required for some of the double mutant phenotypes. To test this, the ING domain coding sequence from MtING1 and MtING2 was cloned and fused to a recombinant N-terminal tag sequence (TRX or MBP) for expression in E. coli . Purified MtING-ING domain protein (MtING ING ) was then tested for oligomerization in vitro using SEC-MALLS analysis. When analysed alone, recombinant TRX:MtING1 ING or MBP:MtING2 ING eluted with a predicted weight-average molecular weight (MW) consistent with monomeric proteins (Fig. 3 F/G). Furthermore, an equimolar mixture of both proteins eluted as two separate peaks with each peak having a MW consistent for two independent monomeric proteins (Fig. 3 H). Thus, the ING domain of the MtING proteins does not dimerise in solution either as homo- or hetero-dimers. In summary, while both ING proteins are located in the plant cell nucleus, the SEC-MALLS analysis indicated that the MtING proteins did not interact to form either homodimers or heterodimers in solution in vitro . Gene expression analyses in the Mting1-7 Mting2-2 double knockout mutant is consistent with its poor growth and non-flowering phenotypes To further investigate the molecular basis of the strongly abnormal phenotypes of the Mting1-7 Mting2-2 double knockout mutant, we analysed leaf and shoot apex gene expression in the double mutant, other Mting mutants and WT in VLD by RNA-seq and RT-qPCR (Tables S1-S5, Fig. 4 ). As expected from the strong mutant phenotypes, gene expression in the Mting1-7 Mting2-2 double knockout mutant differed the most from WT out of all the mutant genotypes. It was strongly separated from all other genotypes in the Principle Component Analysis (PCA) (Fig. 4 A) with thousands of differentially expressed genes (DEGs) compared to WT; 5189 and 3017 upregulated genes and 3064 and 1427 down regulated genes, in leaves and shoot apices respectively (Table S2 , Fig. 4 B). The Mting2-2 single knockout mutant also separated distinctly from WT in the PCA analysis, and had the next highest number of DEGs; 2106 and 660 upregulated and 758 and 326 genes down regulated in leaves and shoot apices respectively compared to WT (Table S2 , Fig. 4 A/B). This is consistent with the abnormal growth and developmental phenotypes of Mting2-2 . In contrast, the Mting1-7 single knockout mutant clustered separately from WT in apex, but not in the leaves, and had fewer DEGs; 504 and 159 upregulated and 250 and 143 down regulated genes in leaves and shoot apices respectively compared to WT (Table S2 , Fig. 4 B). The remaining Mting single and double PHD mutants, clustered with WT in both the leaf and shoot apex samples in the PCA. This included the Mting1-1 Mting2-11 double PHD finger mutant with 584 and 140 up regulated genes and 124 and 41 downregulated genes in the leaf and shoot apex respectively. The most strongly down-regulated gene (Table S2 , S4) in Mting2 single and double mutants relative to WT, in shoot apices and/or leaves, was a candidate histone acetyltransferase gene Medtr5g017020. The predicted encoded protein is of the HAT-KAT11 superclass, in the p300/CBP HAT family (Table S2 , S4). Interestingly, a second, histone acetylation related gene, Medtr5g085310 (Table S2 ), was strongly down regulated in all Mting2 single and double mutants in both tissues. This gene encodes a protein of the HAT-KAT11 superclass in the Zf-TA2 family. Amongst the most strongly up-regulated genes, we noted that in most Mting2 mutants in leaves, these included stress-related genes encoding heat shock protein 70 (Table S2 , S4) and a MYB/SANT domain gene (Table S2 ). Given that the Mting1-7 Mting2-2 double knock out mutant did not produce flowers, and two other Mting mutants had delayed flowering, we then examined the expression of candidate Medicago flowering regulators [ 27 ] in these mutants in RNA-seq and by RT-qPCR (Table S2 , S4, Fig. 4 C/E). In the Mting1-7 Mting2-2 double knockout mutant many MADs genes involved in flowering were expressed at lower levels in the RNA-seq in shoot apices relative to WT. These included the inflorescence meristem identity genes MtAP1 and MtAP1b , the I2 meristem identity gene MtFULc and paralogs MtFULa-b as well as MtSEP1,4 and MtSOC1a-c. Conversely, candidate floral repressors such as SVP- like genes and MtTEM1 and MtTEM2 were elevated in the mutant. Similarly, further analysis of expression of 21 candidate flowering genes by RT-qPCR (Fig. 4 E) revealed that 15 of them were significantly differentially expressed (11 down and 4 up) in the Mting1-7 Mting2-2 double knockout mutant relative to WT. The down regulated genes included the MADS genes above, as well as the potent floral activator MtFTa1 and paralog MtFTb2 , and MtTFL1a which promotes primary inflorescence identity. Up regulated genes included the candidate floral repressors MtBFT, MtTFL1c and MtSVPc. RNA-seq of these genes generally followed the same pattern (Fig. 4 E). The Mting2-2 single knockout mutant showed strongly delayed flowering. It had reduced expression of MtFTa1 and the MADS floral activators and elevated expression of candidate repressors in the RNA-seq as previously reported [ 27 ]. The Mting1-1 Mting2-11 double PHD mutant had a mild delay to flowering. Amongst the MADs box genes, this correlated with down regulation of the important flowering regulator MtAP1 and MtSEP4 compared to WT. On the other hand, the Mting1-7 single knockout mutant flowered similarly to WT and had a similar expression of candidate flowering regulators compared to WT. Since the strong mutant phenotypes and abnormal gene expression of the Mting knockout mutants compared to WT indicated major disruption to regular biological processes, we then carried out a Gene Ontology enrichment analysis for the genes up and down regulated in the Mting1-7 Mting2-2 double knockout mutant and other Mting mutants compared to WT (Table S3 ). We saw that the differentially expressed genes in Mting1-7 Mting2-2 were enriched in many biological processes. In the upregulated genes these included pathways related to plant signalling and stress such as defence and biotic stimulus responses, consistent with the upregulation of the heat shock protein 70. Down regulated enriched processes included photosynthesis, chloroplast-related and metabolism, which was consistent with the very poor growth of the mutant. In the Mting2-2 single knockout mutant, up-regulated enriched pathways in the leaf included core processes such as rRNA processing, while in the shoot apex, upregulated genes were enriched for core processes such as transcription by RNA Polymerase III and in plant responses to biotic stimuli and defence chemical metabolism, like the Mting1-7 Mting2-2 double knock out mutant. Downregulated genes in Mting2-2 in both tissues had similarities to the Mting1-7 Mting2-2 double knockout mutant with enrichment in pathways related to photosynthesis and metabolism. The Mting1-7 mutant had a different profile with upregulated genes in the apex showing enrichment in processes including cellular amide metabolic processes, while in leaf there was enrichment in lipid metabolic processes. There was enrichment in amine biosynthetic processes in apex down regulated genes but no enrichment amongst the down regulated genes in the leaf. Although there have been no described mutants, AtING2 has been shown to interact with components of the NuA4 acetyltransferase complex [ 28 – 31 ]. Furthermore, examination of the NuA4 scaffold protein Enhancer of Polycomb-Like 1 (AtEPL1) double a and b mutant ( Atepl1 ) showed that the plants were small, pale and had a downregulation in genes related to the chloroplast [ 30 , 31 , 52 ]. This is quite similar to the phenotype and differentially expressed genes we saw in the Mting2-2 single knockout and Mting1-7 Mting2-2 double knockout mutants (Table S3 , Fig. 1 ). Therefore, we examined if Medicago homologs of a list of gene targets of NuA4 in Arabidopsis were differentially expressed in the Mting1-7 Mting2-2 double knockout mutant and respective single mutants. The Arabidopsis NuA4 targeted genes were defined previously by a loss of H4K5 acetylation and downregulation in the mutant compared to WT, as determined by ChIP-seq and RNA-seq [ 31 ]. Interestingly, in the Mting1-7 Mting2-2 double knockout mutant and respective single mutants, there was a marked down regulation of the selected genes in the apex tissue, with > 87% of the genes below a log2( Mutant TPM / WT TPM ) of 0 (Table S5 , Fig. 4 D). The magnitude of the movement was also greater in the double mutant, with a median value of -0.72 compared to -0.38 and − 0.44 in the Mting1-7 and Mting2-2 single mutants, respectively. This is opposite to the general pattern of all apex DEGs, which were majority upregulated in Mting2-2 and Mting1-7 Mting2-2 or neutral in Mting1-7 (Table S3 , Fig. 4 B). There was a similar but weaker pattern in the leaves, with log2( Mutant TPM /WT TPM ) median values of -0.25, -0.20 and − 0.33 in the Mting1-7 , Mting2-2 and Mting1-7 Mting2-2 mutants respectively. Overall, this indicates that the MtINGs promote the expression of Medicago homologs of the NuA4 Arabidopsis target genes in WT. Conversely, none of the PHD finger mutants skewed in either direction (Table S5 , Fig. 4 D), suggesting that the PHD finger does not contribute to the regulation of this subset of genes. Discussion The ING gene family is an important group of eukaryotic genes that are involved in the epigenetic regulation of a broad range of cellular processes such as gene regulation, apoptosis, senescence, cell cycle and rRNA synthesis [ 18 , 49 ]. Despite this, relatively little is known about the genetic function of the two plant ING genes, ING1 and ING2 genes, especially in regards to the regulation of flowering time which is critical for crop productivity. Analysis of the Mting1-7 Mting2-2 double knockout mutant indicates combined essential roles of the two MtING genes in flowering To assess the combined role of the Medicago ING genes MtING1 and MtING2 in flowering time and growth we generated Mting double knockout mutants. The late flowering, short stature Mting2-2 mutant was crossed to the WT-like Mting1-7 mutant. The resulting Mting1-7 Mting2-2 double knockout mutants were even more dwarfed, did not maintain outgrowing branches and never flowered. Previous genetic studies of other key Medicago flowering genes have shown that Mtfta1 Mtfda double mutants and Mtsoc1a-c triple mutants are also non-flowering [ 13 , 14 ]. Both of these mutants produced one lateral structure at the leaf axil indicative of remaining vegetative [ 13 , 14 ]. Because the branches of Mting1-7 Mting2-2 mutants withered shortly after initiating, we could not determine the lateral structures in the axil. However, gene expression analysis in the shoot apex showed that key activators upstream of the floral transition including MtFTa1 and all three MtSOC1 genes, and markers of Medicago compound inflorescence meristem identity including MtAP1, MtTFL1a and MtFULc genes were significantly downregulated compared to WT (Fig. 4 ). Furthermore, the putative flowering repressors MtSVPc , MtTFL1c and MtBFT were upregulated. In addition, Mting1-7 Mting2-2 showed > 2 times as many differentially expressed genes amongst candidate flowering gene lists than other Mting mutants, including the late flowering Mting2-2 single mutant. The combined misexpression of some of these genes, together with the greatly reduced MtTFL1a expression and elevated MtSVPc expression compared to the Mting2-2 single mutant, likely contributed to the double knock out mutant remaining vegetative. In summary, the Mting1-7 Mting2-2 double knockout mutant is highly dwarfed and is unable to transition to flowering. This is overall strikingly different from the single mutant parents, Mting2-2 with delayed flowering and small stature and Mting1-7 which appears similar to WT, indicating combined essential roles of the two MtING genes in flowering. Combined MtING gene effects on growth and architecture Beyond flowering time, the strong and diverse phenotypes of the Mting1-7 Mting2-2 double knockout mutant including great reduction in plant height, changes to compound leaf morphology, loss of branching and large global changes to gene expression compared to WT and the single mutant parents, suggests that the two MtING genes have a combined strong pleiotropic effect on gene expression and plant development. Biochemical analyses in Arabidopsis suggested that AtING2 is a scaffold component of the NuA4 acetyltransferase complex with roles in promoting chloroplast development and photosynthesis [ 30 , 31 , 35 , 52 ]. Similarly, photosynthesis and chloroplast-related processes were enriched amongst the down regulated genes in the Mting2-2 single mutant and in the Mting1-7 Mting2-2 double knockout mutant (Table S3 ). There was an overall reduction in the level of expression of Medicago homologs of Arabidopsis NuA4 target genes particularly in the shoot apex in the Mting1-7 , Mting2-2 and Mting1-7 Mting2-2 double mutant compared to WT, with a greater magnitude of reduction in the double mutant (Fig. 4 D). Interestingly, stress response processes were enriched in the upregulated genes in Mting2-2 and Mting1-7 Mting2-2. This is also seen for NuA4 mutants such as Atepl1 [ 31 ]. These results are overall consistent with the idea that MtING1 and MtING2 may regulate gene transcription through histone modification such as through NuA4 action. However, Arabidopsis AtING2 can also bind to Histone Deacetylase Complex 1 (HDC1) [ 53 ] a possible component of plant histone deacetylase machinery [ 54 ] indicating that MtING functions extend beyond NuA4. This is supported by the fact that there were many more up regulated DEGs than down regulated in Mting2-2 and Mting1-7 Mting2-2 mutants compared to WT (Table S2 , Fig. 4 B). This indicates that the MtINGs overall act as a repressor of gene expression rather than an activator [ 27 ]. Analysis of the Mting1-1 Mting2-11 double PHD mutant indicates that the PHD fingers are not essential for MtING function in flowering and development. Our previous work with Mting2 PHD finger single mutants indicated that unexpectedly the conserved PHD finger was not essential for its function, because these single mutants grew and flowered similarly to WT [ 27 ]. To test if the loss of the MtING2 PHD finger was being compensated for by MtING1, we made the Mting1-1 Mting2-11 PHD double mutant. Apart from a strong loss of trichomes, the double mutant showed only a mild delay to flowering time and weak effects on development and gene expression. Therefore, the PHD fingers of both MtINGs do not appear to contribute strongly to the biological function of the MtING proteins in Medicago. Conclusions Our work indicates that MtING1 and MtING2 have overlapping and complementary functions with a combined essential role in the transition to flowering in floral inductive VLD conditions and in growth and development. However, interestingly despite their conservation, the PHD fingers of the MtINGs are not critical for MtING function in flowering and growth. This implicates the other conserved domain, the N-terminal ING domain, as perhaps the most important element for MtING function. Future work should investigate whether the MtINGs are components of chromatin modifying complexes such as a Medicago NuA4 acetyltransferase complex and what genes might be direct targets of this complex. More broadly, based on the foundations provided by our work, it will be important to investigate the genetic functions of the INGs in growth, development and flowering in other plants. Declarations Ethics approval and consent to participate. “Not applicable”. Clinical trial number: “Not applicable”. Consent for publication “Not applicable”. Availability of data and materials. The datasets generated and/or analysed during the current study are available in the GEO repository (GSE277907). Competing interests. “Not applicable”. The authors declare that they have no competing interests. Funding. This work was funded by the Royal Society of New Zealand Te Apārangi through a Marsden grant awarded to J.P (contract 17-UOA-075, used to support M.M-S). Authors' contributions. MMS generated the Mting double mutants, phenotyped the plants, carried out the nuclear localization and SEC-MALLS with DG. LZ performed plant crossing, RNA isolation and RT-qPCR. 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List of primers used SupplementaryTable2.DifferentiallyexpressedgenesinMtingmutants.xlsx Supplementary Table 2. Differentially expressed genes in the apex and leaf of all Mting mutants compared to WT R108, selected based on a cut-off of log2fold-change ≥ 1 or ≤ -1 and adjusted p-value ≤ 0.05. SupplementaryTable3.GeneOntologyanalysisinMtingmutants.xlsx Supplementary Table 3. Gene ontology analysis of differentially expressed genes in all Mting mutants. Top 30 pathways from GO categories Biological Process are presented. Supplementarytable4.Expressionofcandidategenes.xlsx Supplementary Table 4. Expression of candidate differentially expressed genes identified from the apex and leaf of all Mting mutants in RNA-seq (a), the Cheng list [13] (b), and candidates validated by RT-qPCR (c). SupplementaryTable5ExpressionofMedicagohomologsofArabidopsisgenestargetedbyNuA4.xlsx Supplementary Table 5. Expression of Medicago homologs of Arabidopsis genes targeted by NuA4 from Bieluszewski et al. [31] Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 05 Nov, 2024 Editor assigned by journal 25 Oct, 2024 Submission checks completed at journal 25 Oct, 2024 First submitted to journal 22 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5314612","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374308219,"identity":"9dd6c240-e940-4a15-898d-3698fd768bce","order_by":0,"name":"Matthew Mayo-Smith","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Mayo-Smith","suffix":""},{"id":374308221,"identity":"83269d5f-a5fe-4479-905e-d43c57d9bfa6","order_by":1,"name":"Axel Poulet","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Axel","middleName":"","lastName":"Poulet","suffix":""},{"id":374308223,"identity":"338bbda1-410d-4b1b-94fa-7d9a8747b0e7","order_by":2,"name":"Lulu Zhang","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Lulu","middleName":"","lastName":"Zhang","suffix":""},{"id":374308225,"identity":"e33cf5ff-3baa-4fa1-8356-611dfe9651b1","order_by":3,"name":"Yongyan Peng","email":"","orcid":"","institution":"The New Zealand Institute for Plant and Food Research Limited (Plant \u0026 Food Research) Mt Albert","correspondingAuthor":false,"prefix":"","firstName":"Yongyan","middleName":"","lastName":"Peng","suffix":""},{"id":374308226,"identity":"de9df178-dbbd-460c-8fb4-14d45f424805","order_by":4,"name":"David Goldstone","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Goldstone","suffix":""},{"id":374308227,"identity":"ea2df6a0-003b-4c4b-a8d8-6811a7c6d3a4","order_by":5,"name":"Joanna Putterill","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie3PPWrDMBTA8WcEyuLYqyCluYKMwXXA+CwuAk+hpUvJ0MFBoExpryQT8JS2q7v1AB6apbRDP56MR7npmEH/wTISP6QH4HKdYF41/NAJWb/iygP8sKNEF0iIJ7kh9BjpMwSIp9i/CKmmzezwkV0HSFafKo/p5LFu4S6Hi1CPPCwomS7KhULyslUiof6VSKERsKjGiJ8g2XGcRbVTpTMKy4QB1cDr6i/y05ObL0PCDsk3kp19loHonhC8JaEMb/EUkmaEyECk+1IYImdnTyKmrIvTy3vh872dRJtt3a6ynM83sj50t3n0EC6j9u09P+fP9vEj6du2CwDrvmk+fuRyuVyuoV+Y+VD9bVJqOQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Auckland","correspondingAuthor":true,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Putterill","suffix":""}],"badges":[],"createdAt":"2024-10-22 23:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5314612/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5314612/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06432-x","type":"published","date":"2025-04-01T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68432523,"identity":"ac0c5cb1-6a7c-4c5f-9826-d85dad3e4f5e","added_by":"auto","created_at":"2024-11-07 08:09:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2970062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMting1-7 Mting2-2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e double knockout mutants are very small and never flower. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eFlowering time in days to flowering and (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003enodes to flowering in WT, \u003cem\u003eMting1-7\u003c/em\u003e, \u003cem\u003eMting2-2\u003c/em\u003e, and \u003cem\u003eMting1-7 Mting2-2 \u003c/em\u003ein VLD. (\u003cstrong\u003eC-H\u003c/strong\u003e)\u003cstrong\u003e. \u003c/strong\u003eGraphs showing six morphological traits; the distance from the monofoliate to furthest trifoliate (plant spread; \u003cstrong\u003eC\u003c/strong\u003e), total number of compound leaves (\u003cstrong\u003eD\u003c/strong\u003e), width (\u003cstrong\u003eE\u003c/strong\u003e), and height (\u003cstrong\u003eF\u003c/strong\u003e) of the largest terminal leaflet, primary axis height (\u003cstrong\u003eG\u003c/strong\u003e), and percentage of atypical (non-trifoliate) compound leaves on the plant (\u003cstrong\u003eH\u003c/strong\u003e) after 21 days in VLD. Data is shown as a boxplot with median and interquartile range. Statistical significance was determined using a Wilcoxon test (\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003e B\u003c/strong\u003e,\u003cstrong\u003e H, I, J\u003c/strong\u003e; 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\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC-G\u003c/strong\u003e). Groups with different letters are statistically different from each other. (\u003cstrong\u003eI\u003c/strong\u003e) The total chlorophyll concentration in µg per g of fresh weight and ratio of chlorophyll a to b (\u003cstrong\u003eJ\u003c/strong\u003e) in the compound leaves of 33 day old WT, \u003cem\u003eMting1-7\u003c/em\u003e and \u003cem\u003eMting2-2\u003c/em\u003e or 233 day old \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e 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. (\u003cstrong\u003eK\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePhotographs\u003cstrong\u003e \u003c/strong\u003efrom above of WT, \u003cem\u003eMting1-7\u003c/em\u003e, \u003cem\u003eMting2-2\u003c/em\u003e, and \u003cem\u003eMting1-7 Mting2-2 \u003c/em\u003eafter 14 days in VLD or from the side after 33 days in VLD (\u003cstrong\u003eL\u003c/strong\u003e). (\u003cstrong\u003eM\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e mutant after 75 days and 125 days in VLD. (\u003cstrong\u003eN\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eComparison between trichomes on a terminal leaflet adaxial surface in WT and \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e. Scale bars are 1 cm unless labelled otherwise.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/b3554342703b5bcce21a5869.png"},{"id":68432519,"identity":"a33b1a02-ecde-4d5d-80d4-8918c697810e","added_by":"auto","created_at":"2024-11-07 08:09:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1849518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMting1-1 Mting2-11 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edouble PHD\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutants are smaller and later flowering than WT\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eFlowering time in days to flowering and (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003enodes to flowering in WT, \u003cem\u003eMting1-1\u003c/em\u003e, \u003cem\u003eMting2-11\u003c/em\u003e, and \u003cem\u003eMting1-1 Mting2-11 \u003c/em\u003ein VLD.\u003cstrong\u003e (C\u003c/strong\u003e) Graphs showing the total number of compound leaves and (\u003cstrong\u003eD\u003c/strong\u003e) percentage of atypical (non-trifoliate) compound leaves on the plant after 21 days in VLD. (\u003cstrong\u003eE\u003c/strong\u003e) 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 (\u003cstrong\u003eA-C\u003c/strong\u003e) (Groups with different letters are significantly different) or Wilcoxon test (\u003cstrong\u003eD\u003c/strong\u003e,\u003cstrong\u003e E\u003c/strong\u003e; p-value with Bonferroni correction: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). (\u003cstrong\u003eF\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePhotograph showing WT, \u003cem\u003eMting1-1\u003c/em\u003e, \u003cem\u003eMting2-11\u003c/em\u003e and \u003cem\u003eMting1-1 Mting2-11 \u003c/em\u003eplants.\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003eG\u003c/strong\u003e) Typical compound leaf and (\u003cstrong\u003eH\u003c/strong\u003e) the adaxial leaf surface comparing trichomes on WT and \u003cem\u003eMting1-1 Mting2-11 \u003c/em\u003eafter 32 days in VLD. Scale bars are 1 cm unless labelled otherwise.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/96c9628bfdbc0778b058434b.png"},{"id":68432520,"identity":"4082a108-5010-4bc2-94c9-6fbed0704fda","added_by":"auto","created_at":"2024-11-07 08:09:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":572679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMtING proteins are nuclear localized but do not interact \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eto form dimers.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSchematic diagram showing the location of the nuclear localization signal (NLS) domain in HsING4. (\u003cstrong\u003eB\u003c/strong\u003e) Protein sequence alignment of the human HsING4 NLS domain and the homologous Medicago ING regions and (\u003cstrong\u003eC\u003c/strong\u003e) end of the PHD finger. The HsING4 NLS (black line) and nucleolar localization sequence (NoLS, grey lines) identified by Tsai \u003cem\u003eet al\u003c/em\u003e. [48] are shown. The numbering is based on the relative position within ING4. (\u003cstrong\u003eD\u003c/strong\u003e) A table showing potential predicted NLS peptides using NLStradamus [38]. (\u003cstrong\u003eE\u003c/strong\u003e) Confocal microscopy images from tobacco explants co-transformed with \u003cem\u003eNLS:mCherry \u003c/em\u003e(red channel) and either \u003cem\u003eeGFP\u003c/em\u003e, \u003cem\u003eMtING1:eGFP \u003c/em\u003eor\u003cem\u003e MtING2:eGFP\u003c/em\u003e (green channel) overexpression constructs. Photographs are representative of two separate experiments each with three independent transformations. Scale is 100 µM. (\u003cstrong\u003eF\u003c/strong\u003e) SEC-MALLS was used to analyse if tagged MtING proteins dimerised \u003cem\u003ein vitro\u003c/em\u003e. Graph showing the elution profile for TRX-tagged MtING1 ING domain (TRX:MtING1\u003csub\u003eING\u003c/sub\u003e) and (\u003cstrong\u003eG\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eMBP-tagged MtING2 ING domain (MBP:MtING2\u003csub\u003eING\u003c/sub\u003e) recombinant protein at a concentration of ~4.25 mg/ml (289 µM or 62.8 µM for ING1 or ING2, respectively) or (\u003cstrong\u003eH\u003c/strong\u003e) 1:1 equimolar (51.4 µM) mixture of MBP:ING2\u003csub\u003eING\u003c/sub\u003e and TRX:MtING1\u003csub\u003eING\u003c/sub\u003e. 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\u003csub\u003eING\u003c/sub\u003e protein is indicated by a dashed grey line.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/b1696a269d4d925d9dcdc8a1.png"},{"id":68432522,"identity":"2abcd406-7277-40ec-94ea-4349eecda9a9","added_by":"auto","created_at":"2024-11-07 08:09:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":350394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMting1-7 Mting2-2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is consistent with non-flowering and has similarities to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtepl1\u003c/strong\u003e\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) PCA plots for apex and leaf tissue RNA-seq data from the single parental \u003cem\u003eMting \u003c/em\u003emutant lines, double \u003cem\u003eMting\u003c/em\u003e mutants and WT. Three biological replicates were harvested for each genotype. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eA\u003cstrong\u003e \u003c/strong\u003eheat map of the log2 fold change obtain with the deseq2 analysis of all up and down differentially expressed genes in \u003cem\u003eMting1-7\u003c/em\u003e, \u003cem\u003eMting2-2\u003c/em\u003e and \u003cem\u003eMting1-7\u003c/em\u003e \u003cem\u003eMting2-2\u003c/em\u003e relative to WT and (\u003cstrong\u003eC\u003c/strong\u003e) 93 selected candidate genes extracted by Cheng \u003cem\u003eet al.\u003c/em\u003e [13] in all mutants relative to WT. The data is represented as the log2 of \u003cem\u003eMutant\u003c/em\u003e\u003csub\u003eTPM\u003c/sub\u003e/WT\u003csub\u003eTPM\u003c/sub\u003e. (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eBoxplot of the log2(\u003cem\u003eMutant\u003c/em\u003e\u003csub\u003eTPM\u003c/sub\u003e/WT\u003csub\u003eTPM\u003c/sub\u003e) in all \u003cem\u003eMting \u003c/em\u003emutants 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 \u003cem\u003eAtepl1\u003c/em\u003e NuA4 mutant and WT [31]. (\u003cstrong\u003eE\u003c/strong\u003e) RT-qPCR analysis (left panel) and RNA-seq (right panel) of 21 candidate flowering genes in \u003cem\u003eMting \u003c/em\u003emutant 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.\u003c/p\u003e\n\u003cp\u003e≤\u003c/p\u003e","description":"","filename":"Fig4v2.png","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/c24ade97d2e3076ca8284e8f.png"},{"id":80082389,"identity":"7a19e8f9-3729-4474-892d-a41bcbec8584","added_by":"auto","created_at":"2025-04-07 16:08:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6562100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/2501a303-290b-4354-95f6-dc856342104a.pdf"},{"id":68432521,"identity":"8cff10f3-33ff-4123-b32a-9aef23db07df","added_by":"auto","created_at":"2024-11-07 08:09:53","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e. List of primers used\u003c/p\u003e","description":"","filename":"SupplementaryTable1.Listofprimersused.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/d68c98fef442b64889aaf6cb.xlsx"},{"id":68432524,"identity":"127ac575-fc03-4606-8e3a-d6abcb0b9cb9","added_by":"auto","created_at":"2024-11-07 08:09:53","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1720057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2. \u003c/strong\u003eDifferentially expressed genes in the apex and leaf of all \u003cem\u003eMting \u003c/em\u003emutants compared to WT R108, selected based on a cut-off of log2fold-change ≥ 1 or ≤ -1 and adjusted p-value ≤ 0.05.\u003c/p\u003e","description":"","filename":"SupplementaryTable2.DifferentiallyexpressedgenesinMtingmutants.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/7d0467e9edc308b3ba69a2ce.xlsx"},{"id":68432527,"identity":"955d7122-88d4-480c-9db1-2c328e6be1e7","added_by":"auto","created_at":"2024-11-07 08:09:53","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":104582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3. \u003c/strong\u003eGene ontology analysis of differentially expressed genes in all \u003cem\u003eMting \u003c/em\u003emutants. Top 30 pathways from GO categories Biological Process are presented.\u003c/p\u003e","description":"","filename":"SupplementaryTable3.GeneOntologyanalysisinMtingmutants.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/529e4ace88830e362c24ce27.xlsx"},{"id":68432967,"identity":"88073b80-ffe5-45c6-a218-e774d356ac60","added_by":"auto","created_at":"2024-11-07 08:17:53","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":91277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 4. \u003c/strong\u003eExpression of candidate differentially expressed genes identified from the apex and leaf of all \u003cem\u003eMting \u003c/em\u003emutants in RNA-seq (a), the Cheng list [13] (b), and candidates validated by RT-qPCR (c).\u003c/p\u003e","description":"","filename":"Supplementarytable4.Expressionofcandidategenes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/a84a424d472f5bdb4a1440b5.xlsx"},{"id":68432968,"identity":"56775e71-9f6d-4f7a-a79d-9daece1fd46e","added_by":"auto","created_at":"2024-11-07 08:17:53","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":114974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 5. \u003c/strong\u003eExpression of Medicago homologs of Arabidopsis genes targeted by NuA4 from Bieluszewski \u003cem\u003eet al. \u003c/em\u003e[31]\u003c/p\u003e","description":"","filename":"SupplementaryTable5ExpressionofMedicagohomologsofArabidopsisgenestargetedbyNuA4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314612/v1/0836af4f0f2ecf433707fe87.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Medicago Mting1 Mting2 double knockout mutants are extremely dwarfed and never flower implicating essential MtING functions in growth and flowering","fulltext":[{"header":"Background","content":"\u003cp\u003eThe timing of flowering is a major factor in successful plant adaptation and crop productivity, including in the legume family which is the second most economically important plant group after the cereals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Like winter annual Arabidopsis, the temperate model legume \u003cem\u003eMedicago truncatula\u003c/em\u003e (Medicago) is induced to flower by extended cold (vernalization, V) followed by warm long day (LD) conditions (VLD) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, strikingly in Medicago there is no CONSTANS that promotes flowering in LD, or FLOWERING LOCUS C to repress flowering like in Arabidopsis [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, a predicted Polycomb complex PRC2 component MtVRN2 has a different function in flowering in Medicago to Arabidopsis, repressing the important \u003cem\u003eFLOWERING LOCUS T (FT)\u003c/em\u003e-like gene \u003cem\u003eMtFTa1\u003c/em\u003e prior to vernalization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite this, other Medicago homologs appear to promote or repress flowering in a similar manner to Arabidopsis, such as the Medicago floral activators \u003cem\u003eMtFTa1\u003c/em\u003e, \u003cem\u003eSUPPRESSOR OF CONSTANS 1\u003c/em\u003e (\u003cem\u003eSOC1\u003c/em\u003e) \u003cem\u003eMtSOC1a\u003c/em\u003e, the photoreceptor MtPHYTOCHROME A which strongly promotes flowering in LD and \u003cem\u003eMtFE\u003c/em\u003e, or repressors such as MtCYCLING DOF FACTORS [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, the FT-FLOWERING LOCUS D (FD) complex is crucial for flowering in Medicago as double \u003cem\u003eMtfta1 Mtfda\u003c/em\u003e mutants never flower [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Gene edited \u003cem\u003eMtsoc1a Mtsoc1b Mtsoc1c\u003c/em\u003e triple mutants also do not transition to flowering, implicating a broader role for the duplicated \u003cem\u003eMtSOC1\u003c/em\u003e-like genes in Medicago than \u003cem\u003eSOC1\u003c/em\u003e in Arabidopsis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eINHIBITOR OF GROWTH\u003c/em\u003e (\u003cem\u003eING\u003c/em\u003e) genes are found in most eukaryotes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. First identified as a tumour suppressor in humans [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], they have since been studied in other organisms such as yeast and are known to be involved in the regulation of gene transcription and development [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The genes encode proteins with two conserved domains, a N-terminal ING domain and a C-terminal plant homeodomain (PHD) finger. The ING domain has been shown to facilitate protein-protein interactions, including to recruit activator histone acetyltransferase or repressor histone deacetylase complexes to genes influencing their transcription [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For example, in yeast the YNG2 ING protein is a part of the NuA4 histone acetyltransferase [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The C-terminal PHD domain is a Cys4-His Cys3 zinc finger that binds to the chromatin mark H3K4me3 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany plants, including Medicago and Arabidopsis, have two \u003cem\u003eING\u003c/em\u003e genes; \u003cem\u003eING1\u003c/em\u003e and \u003cem\u003eING2\u003c/em\u003e [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. They encode proteins which are highly similar between plant species, with the Arabidopsis and Medicago proteins sharing 68% (ING1) to 80% (ING2) sequence identity, while only being ~\u0026thinsp;30% identical between ING1 and ING2 within species [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The Arabidopsis ING proteins are present in the nucleus and can bind H3K4me3 peptides \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, binding studies and mass spectrometry indicate that Arabidopsis ING2 may be a part of a NuA4 complex, functioning as a scaffold in the Piccolo catalytic module [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNo Arabidopsis \u003cem\u003eAting\u003c/em\u003e mutant studies have been published yet, but recently we showed in Medicago that gene-edited \u003cem\u003eMting2\u003c/em\u003e single knockout mutant plants were pale in colour and dwarfed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. They also had reduced \u003cem\u003eMtFTa1\u003c/em\u003e expression and delayed flowering, especially in floral inductive VLD conditions compared to wild type (WT) plants [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Analysis of a range of different gene-edited \u003cem\u003eMting2\u003c/em\u003e mutant plants indicated that an intact MtING2 ING domain was important for WT-like growth, development and flowering. However, unexpectedly, we saw that mutations that affected the C-terminal PHD finger of MtING2, but left the ING domain intact, had little effect on plant growth and development. Gene edited \u003cem\u003eMting1\u003c/em\u003e single knockout mutants also grew and flowered similar to WT [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere we make and characterise the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant which has striking abnormal growth phenotypes and never flowers, indicating genetic redundancy as well as complementary functions. We identify differentially expressed genes by RNA-seq and RT-qPCR and compare these to Arabidopsis genes identified as potential NuA4 histone acetyltransferase targets [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We also investigate possible genetic redundancy of the PHD finger encoded by the \u003cem\u003eING\u003c/em\u003e genes by evaluating the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutant.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePlant material and growth.\u003c/b\u003e Wild type Medicago R108_C3 (WT) was used in this study [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The \u003cem\u003eMting1\u003c/em\u003e and \u003cem\u003eMting2\u003c/em\u003e single mutants: \u003cem\u003eMting1-1, Mting1-7, Mting2-2\u003c/em\u003e and \u003cem\u003eMting2-11\u003c/em\u003e were previously reported by Jaudal \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eMting1 Mting2\u003c/em\u003e double mutants were generated by crossing in this study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To generate \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant and the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD finger mutant, the respective \u003cem\u003eMting1\u003c/em\u003e and \u003cem\u003eMting2\u003c/em\u003e mutant plants were manually cross-pollinated as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The combination of \u003cem\u003eMting\u003c/em\u003e alleles in the subsequent segregating F1 and F2 progeny was confirmed using allele specific genomic PCR (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescription of \u003cem\u003eMting1\u003c/em\u003e and \u003cem\u003eMting2\u003c/em\u003e single and double mutants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epredicted mutation effect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003emutation description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epredicted length (aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ephenotypes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMting1-7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epredicted knockout\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edeletion causing a frame shift, encoding highly truncated protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWT-like*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMting2-2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epredicted knockout\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elarge deletion causing a frame shift, encoding highly truncated protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esmall, late flowering, pale*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMting1-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePHD finger deletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elarge deletion, removing encoded PHD finger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWT-like*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMting2-11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePHD finger deletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esmall indels, affecting encoded ING domain loop region and removing PHD finger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWT-like*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003edouble knockout mutant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eas above\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003etiny, no branches, never flowers\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003edouble PHD finger mutant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eas above\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emild developmental and flowering phenotypes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe predicted length of WT MtING1 is 247 amino acids (aa), and MtING2 is 263 aa.\u003c/p\u003e \u003cp\u003e*according to Jaudal \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAll Medicago plants used for this research were grown under VLD which is a floral-inductive condition. To sow plants, seeds were scarified using sandpaper (p600 grit), sterilised in a chlorine solution (Millipore, USA) for 10 minutes and germinated overnight, shaking in water at 15\u0026deg;C in the dark. Seedlings were vernalized for 3 weeks at 4\u0026deg;C on moist filter paper in a petri dish. Vernalized seedlings were planted directly into seed-raising mix (Daltons, NZ) in small 6-cell punnets placed on rockwool mats (Grodan, NE) sub irrigated with hydroponics [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] (without Na\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eSi). The plants were grown in a controlled greenhouse under white fluorescent light (~\u0026thinsp;160\u0026ndash;200 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in long days (16/8 hours light/dark) at 22\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNicotiana tabacum\u003c/em\u003e (tobacco) was used for transient expression of \u003cem\u003e35S:MtING-GFP\u003c/em\u003e, \u003cem\u003e35S-eGFP\u003c/em\u003e and \u003cem\u003e35S:NLS-mCherry\u003c/em\u003e. Seeds were sown at high density directly into seed raising mix and healthy seedlings were transplanted into 0.5 L pots with planting mix (Daltons) watered with hydroponics solution. The tobacco plants were grown under the same conditions as the Medicago.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotypic analysis.\u003c/b\u003e The flowering time of plants was measured by the number of days after planting, and the number of nodes on the primary axis at the emergence of the first floral bud on the plant. For the flowering time data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the sample size (n) ranged from 4 to 24 and in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 8 to 16. All other data is presented at a stated plant age where n\u0026thinsp;=\u0026thinsp;6 to 11 for the data in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, or 8 to 16 for Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The plant spread was measured as the horizontal distance in millimetres from the monofoliate leaf tip to the leaf tip of the furthest branch. The total number of leaves was measured as the total number of fully expanded compound leaves plus the monofoliate leaf on the plant. The length of the primary axis was measured as the distance in millimetres from the base of the monofoliate node to the growth tip. The percentage of atypical leaves was measured as the number of non-trifoliate compound leaves (excluding the monofoliate) on the plant divided by the total leaf number.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll phenotypic data is graphed as a boxplot with median and interquartile range. Statistical significance was tested with a one-way ANOVA and Tukey honest significance test (p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05) or where the data does not satisfy the assumptions of an ANOVA, a Wilcoxon test with Bonferroni correction testing for a difference to WT.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChlorophyll measurements.\u003c/b\u003e Chlorophyll content was measured according to Porra \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] with modification. Chlorophyll was extracted from two leaves from a plant for each of the three biological replicates after 33 days in VLD for WT, \u003cem\u003eMting1-7\u003c/em\u003e and \u003cem\u003eMting2-2\u003c/em\u003e, or three replicates from different leaves on one \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double mutant plant after 233 days. Two of the newest fully expanded compound leaves on the primary axis were harvested, weighed (approximately 100\u0026ndash;150 mg) and frozen in liquid nitrogen. Chlorophyll was extracted from ground tissue using 2\u0026ndash;5 ml of Tris-HCl pH 7 buffered 80% acetone. Samples were processed four at a time and diluted in additional buffered acetone so the absorbance at 663 nm was between 0.5 to 0.8. The absorbance at 646 nm and 663 nm (with correction at 750 nm) was measured using a Cary 4000 spectrophotometer (Agilent, USA). The total chlorophyll per gram of fresh weight and the chlorophyll a to b ratio was calculated according to Porra \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The statistical significance from WT was tested using a t-test assuming unequal variance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescent protein localization assay.\u003c/b\u003e The full-length \u003cem\u003eMtING1\u003c/em\u003e or \u003cem\u003eMtING2\u003c/em\u003e coding sequence was cloned into a pHEX2 vector [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] modified for fusion with eGFP at the C-terminal end of the encoded proteins under the cauliflower mosaic virus constitutive 35S promoter. A control \u003cem\u003e35S:eGFP\u003c/em\u003e was also cloned into pHEX2. A positive nuclear targeted control \u003cem\u003e35S:NLS:mCherry\u003c/em\u003e was also cloned using Golden Gate assembly [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The vectors were transformed independently into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 and cultured. \u003cem\u003eA. tumefaciens\u003c/em\u003e containing the respective \u003cem\u003eGFP\u003c/em\u003e and \u003cem\u003emCherry\u003c/em\u003e vector were mixed in equal parts and resuspended into infection medium (50 mM MES pH 5.6, 2 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 28 mM glucose, 200 \u0026micro;M acetosyringone), and injected into the two newest leaves of 5\u0026ndash;7 leaf stage tobacco plants. Small circular discs were sampled from the infiltrated leaves after three or five days. Transformations were repeated in two independent experiments with three independently transformed plants per experiment.\u003c/p\u003e \u003cp\u003eConfocal laser scanning microscopy images were captured on an Eclipse Ti-E microscope with a 40x S Plan Fluor ELWD objective lens (Nikon, Japan), CSU-X1 confocal scanner unit (Yokogawa, Japan), ALC-500 laser unit and Zyla sCMOS camera (Andor, UK). GFP and mCherry were excited using a laser light wavelength of 488 nm and 561 nm, respectively, and images captured with a 3035B and TX Red 4040B filter (Semrock, USA). For each sample, greater than 100 cells from each leaf disc were observed. Single slice images were processed using NIS-Elements imaging software (Nikon, v4.06.12).\u003c/p\u003e \u003cp\u003ePotential nuclear localization signal (NLS) sites for the MtING proteins were identified using NLStradamus [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The full-length sequences of human ING4, MtING1 and MtING2 were queried using a 4-state HHM static model with 0.6 threshold on the NLStradamus webserver [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eSEC-MALLS.\u003c/b\u003e Size exclusion chromatography (SEC) with multi angle laser light scattering (SEC-MALLS) was used to determine the weight-average molecular weight (MW) and oligomeric state of recombinant MtING ING domain protein in solution. The coding sequence of the ING domain of MtING1 and MtING2, named MtING1\u003csub\u003eING\u003c/sub\u003e (M1-E118) and MtING2\u003csub\u003eING\u003c/sub\u003e (M1-E133), was cloned from WT into a pET-49b(+) vector for N-terminal fusion with either THIOREDOXIN (TRX:MtING1\u003csub\u003eING\u003c/sub\u003e) or MALTOSE BINDING PROTEIN (MBP:MtING2\u003csub\u003eING\u003c/sub\u003e). The induction of recombinant protein expression was the same as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Protein was purified from cell lysate using either nickel immobilised metal affinity chromatography (TRX:MtING1\u003csub\u003eING\u003c/sub\u003e) or amylose affinity chromatography (MBP:MtING2\u003csub\u003eING\u003c/sub\u003e) followed by SEC, and then concentrated to 4.25 mg/mL using an Amicon 3K centrifugal concentrator (Merck, De).\u003c/p\u003e \u003cp\u003ePurified samples (100 \u0026micro;L) were analysed using SEC-MALLS at 25\u0026deg;C with a Dionex UltiMate 3000 HPLC pump (Thermo Fisher, USA), SLD7000 7-angle MALL detector (PSS, De), RI-101 differential refractive index detector (Shodex, De) and Superdex 200 Increase 10/300 GL 24 mL column (General Electric, USA) in MALLS buffer (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.1 mM TCEP, 3 mM azide). The MW was determined using PSS winGPC Unichrom software.\u003c/p\u003e \u003cp\u003e\u003cb\u003ePlant tissue harvesting and RNA extraction.\u003c/b\u003e For RNA-seq and real time reverse transcription quantitative PCR (RT-qPCR), leaf and shoot apex tissue was harvested from plants grown under VLD conditions, 4 h after dawn on day 14 (WT, \u003cem\u003eMting1-7\u003c/em\u003e, \u003cem\u003eMting1-1\u003c/em\u003e, \u003cem\u003eMting2-11\u003c/em\u003e and \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e), day 15 (\u003cem\u003eMting2-2\u003c/em\u003e) and day 21 (\u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e) when the plants had 2\u0026ndash;3 fully expanded compound leaves. Three biological replicates were harvested per tissue type per genotype, each consisting of 1 to 2 fully expanded compound leaves from the same plant, or 3 to 4 primary apices from different plants. Harvested tissue was snap-frozen in liquid nitrogen and homogenised by metal beads in a Geno/Grinder\u0026reg; 2010 (New Jersey, USA). Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, De) according to the user manual. RNA quantity and quality were checked by a NanoPhotometer\u0026reg; N60 (Implen, De) and a Bioanalyzer 2100 (Agilent Technologies, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq and analysis.\u003c/b\u003e The RNA-seq was carried out by Novogene (Hong Kong) with library preparation and sequencing as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Directional strand specific mRNA libraries (poly A enrichment) were prepared and sequenced on the Illumina NovaSeq platform NovaSeq6000, 150 bp paired-end (Novogene, Hong Kong). The FASTQ file read quality was evaluated, and Fastp (v0.21) was used for trimming [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Reads with a quality below a PHRED score of 20 were trimmed from the 3\u0026rsquo;end, and reads\u0026thinsp;\u0026lt;\u0026thinsp;36 bp in length were excluded. The remaining reads were mapped against the Mt4.0v2 transcriptome [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] using Salmon (v0.8.2). DESeq2 (v1.24.0) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] was used for normalization and differential expression analyses. Differentially expressed transcripts were filtered using a cut-off adjusted p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and log2 fold-change\u0026thinsp;\u0026ge;\u0026thinsp;1 or \u0026le;\u0026thinsp;\u0026minus;\u0026thinsp;1. To identify candidate direct target genes of NuA4, we used Blastx [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] against Arabidopsis NuA4 bound and regulated genes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. All the genes with an e-value\u0026thinsp;\u0026lt;\u0026thinsp;e\u0026thinsp;\u0026minus;\u0026thinsp;100 were selected. Genes that were not expressed or only weakly expressed were removed, which gave a list of 289 transcripts (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene expression analysis by RT-qPCR.\u003c/b\u003e The WT and \u003cem\u003eMting1-7 Mting2-\u003c/em\u003e2 RNA samples were treated with DNase (TURBO DNA-\u003cem\u003efree\u003c/em\u003e\u0026trade; Kit, Invitrogen, USA) according to manufacturer's instructions. Template cDNA was synthesised with SuperScript\u0026trade; IV Reverse Transcriptase (Invitrogen) as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Gene expression analysis was performed based on the comparative CT method [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], with modifications [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. ΔCT values were obtained by normalising genes of interest to the reference gene, \u003cem\u003ePROTEIN PHOSPHATASE 2A\u003c/em\u003e (\u003cem\u003ePP2A\u003c/em\u003e, Medtr6g084690). Relative gene expression was calculated using the formula 2\u003csup\u003e\u0026minus;ΔCT\u003c/sup\u003e. Statistical significance between WT and \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e was calculated using the t-test, assuming unequal variance (p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05). RT-qPCR primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMting1-7 Mting2-2\u003c/b\u003e \u003cb\u003edouble knockout mutants have severe growth and developmental defects and never flower\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePreviously, we showed that \u003cem\u003eMting2\u003c/em\u003e single knockout mutations strongly impacted growth and delayed flowering in Medicago, but \u003cem\u003eMting1\u003c/em\u003e single mutants were similar to WT [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To assess the physiological consequences of strong mutations in both \u003cem\u003eMtING\u003c/em\u003e genes, we generated a double knockout mutant by crossing \u003cem\u003eMting1\u003c/em\u003e and \u003cem\u003eMting2\u003c/em\u003e single knockout mutants. The \u003cem\u003eMting1-7\u003c/em\u003e single knockout mutant carried a deletion causing a frameshift in the encoded protein. This led to an altered amino acid (aa) sequence from aa 21 onwards and a truncated protein of 83 aa, compared to 247 aa WT MtING1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The small, pale green and late flowering \u003cem\u003eMting2-2\u003c/em\u003e single knockout mutant was predicted to encode a truncated protein with a deletion of aa 16 to 39 and a final protein of 44 aa, compared to WT MtING2 of 263 aa (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After crossing, we grew F1 plants and then identified homozygous \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double mutant F2 progeny by PCR genotyping (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe double mutants had multiple highly abnormal phenotypes, compared to the single mutant parents and WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As expected, in the parental lines, \u003cem\u003eMting1-7\u003c/em\u003e flowered like WT, while \u003cem\u003eMting2-2\u003c/em\u003e flowered late in floral inductive VLD conditions. Conversely, the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant never produced a floral bud (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA/B). The double mutants continued to grow very slowly, but still had not flowered after six months after which they started to die. The double mutants were severely dwarfed even when compared to the small \u003cem\u003eMting2-2\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK/L). The \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e plants also had significantly reduced plant diameter (plant spread), smaller leaflets and fewer compound leaves compared to \u003cem\u003eMting2-2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-F). The double mutants could not maintain outgrowing branches, because they withered as the plants grew (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Similar to the \u003cem\u003eMting2-2\u003c/em\u003e single mutant parent [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], they had an increase in the percentage of atypical compound leaves and no leaf trichomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH/N). Therefore, this double mutant analysis implicates both \u003cem\u003eMtING\u003c/em\u003e genes as essential for normal development in Medicago, with redundant and complementary roles in the regulation of flowering, growth and plant architecture.\u003c/p\u003e \u003cp\u003eOtherwise, \u003cem\u003eMting1-7\u003c/em\u003e single mutants had a slight increase in plant size when compared to WT in five of the measured morphological traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-G) at 21 days in VLD. This indicates that \u003cem\u003eMting1-7\u003c/em\u003e in an otherwise WT background appears to have a weakly promotive effect on Medicago plant size, unlike \u003cem\u003eMting2-2\u003c/em\u003e mutants which are dwarfed compared to WT. Previously, we reported that \u003cem\u003eMting2\u003c/em\u003e mutants had a pale colour [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To determine if this was the result of changes in chlorophyll levels, we measured the chlorophyll content in the leaves of the mutants compared to WT. \u003cem\u003eMting2-2\u003c/em\u003e mutants had a reduced total chlorophyll amount compared to WT and \u003cem\u003eMting1-7\u003c/em\u003e, but there was no change in the ratio of chlorophyll a to chlorophyll b for any plants indicating that \u003cem\u003eMting2\u003c/em\u003e affects the production of both chlorophylls equally (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI/J). In contrast, the double knockout mutant displayed similar chlorophyll levels to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI/J).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMting1-1 Mting2-11\u003c/b\u003e \u003cb\u003edouble PHD mutants show only mild growth and flowering phenotypes but a strong reduction in leaf trichomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePreviously, our analysis of a range of gene-edited \u003cem\u003eMting2\u003c/em\u003e single mutants indicated that the ING domain of MtING2 was important for normal growth and flowering in Medicago [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. But, unexpectedly, \u003cem\u003eMting2\u003c/em\u003e mutants with changes to or loss of the PHD finger domain grew similarly to WT, indicating that the MtING2 PHD finger domain was not necessary in an otherwise WT background [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs demonstrated above, \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutants had a much stronger abnormal growth and development phenotypes than the parental lines, indicating that \u003cem\u003eMtING1\u003c/em\u003e and \u003cem\u003eMtING2\u003c/em\u003e had overlapping and complementary functions. Thus, we hypothesised that \u003cem\u003eMting\u003c/em\u003e double PHD finger mutants may also exhibit enhanced mutant phenotypes compared to the single PHD finger mutants. To test this, we crossed \u003cem\u003eMting2-11\u003c/em\u003e plants with mutations predicted to only affect the PHD coding sequence and the non-essential loop region of the ING domain and which grew similarly to WT, to \u003cem\u003eMting1-1\u003c/em\u003e plants with mutations predicted to remove large portions of the PHD finger, but not affecting the ING domain and which also grew like WT (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After crossing, we selected the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD finger mutants in the F2 generation by PCR genotyping (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhenotyping of the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) showed that they flowered slightly later in both days and nodes to flowering than WT and the parents (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA/B). The parental lines showed a mild delay to flowering in days, but not in nodes compared to WT. The \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutants still flowered much earlier than the \u003cem\u003eMting2-2\u003c/em\u003e knockout plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA/B). This suggested that both ING PHD fingers made a mild contribution to the promotion of flowering, but had a much weaker effect than the intact ING proteins. In addition, the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutant plants and their compound leaves appeared more compact than WT and the single mutant parental lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF/G), indicating again a mild contribution of both of the MtING PHD fingers on plant growth. Interestingly, the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutants also displayed greatly reduced visual leaf trichome density compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). The reduction in trichomes was similar to the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). This implicates both MtING PHD fingers as vital for normal leaf trichome development.\u003c/p\u003e \u003cp\u003eIn other phenotypes, the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutant and the \u003cem\u003eMting2-11\u003c/em\u003e parent had a similarly slightly shorter primary axis and a higher percentage of atypical compound leaves, compared to WT and \u003cem\u003eMting1-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD/E). This suggested that loss of the MtING2 PHD finger alone was sufficient for this mild dwarfing and change in leaf patterning. Compared to WT, total leaf number was slightly reduced to a similar level in \u003cem\u003eMting1-1\u003c/em\u003e and the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn summary, \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutants had weakly abnormal growth and delayed flowering phenotypes and strong loss of leaf trichomes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMtING proteins localize to the nucleus in tobacco cells but do not dimerize\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHuman ING proteins contain a nuclear localization signal (NLS) in the disordered linker between the PHD finger and N-terminal ING domain and are nuclear localized [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Furthermore, some human ING proteins contain an additional nucleolar localization signal (NoLS) within the NLS or downstream of the PHD finger [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Previously, it was shown that Arabidopsis AtING1 and AtING2 GFP-tagged proteins located to the nucleus in transient expression in protoplasts, although the NLS and NoLS identified in human INGs were not found in these ING proteins [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we first compared the known NLS from human ING4 to the homologous regions of MtING1 and MtING2 and also used \u003cem\u003ein silico\u003c/em\u003e analysis to predict NLS in the Medicago proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). Both of the Medicago INGs contain an enrichment of basic R and K residues in the NLS domain region which is similar to that of the human ING4 NLS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, MtING1, but not MtING2, has a RK rich sequence down downstream of the PHD finger, similar to the second NLS of ING4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This is supported by the \u003cem\u003ein silico\u003c/em\u003e prediction which identified both sites as potential NLS for MtING1. No sites were predicted for MtING2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Therefore, to empirically test if MtING1 and MtING2 are nuclear localised, we generated overexpression constructs designed to express MtING1 and MtING2 translational fusions with C-terminal eGFP and used Agroinfiltration for transient expression in tobacco leaves. An \u003cem\u003eeGFP\u003c/em\u003e construct was infiltrated as a positive GFP control, while a \u003cem\u003eNLS-mCherry\u003c/em\u003e construct was co-transformed with the \u003cem\u003eeGFP\u003c/em\u003e constructs and provided a positive nuclear control. Confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) indicated that both MtING1 and MtING2 fusion proteins are nuclear localized, like the control NLS-mCherry. In contrast the control eGFP was located to both nucleus and cytoplasm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStudies of three of the human ING proteins most closely related to the plant INGs have shown that they form homodimers and heterodimers through the N-terminal ING domain in solution [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Thus it is possible that plant ING proteins are also physically interacting through the ING domain. If the plant ING proteins do form homo or hetero dimers \u003cem\u003ein planta\u003c/em\u003e this could help to explain why mutation of both \u003cem\u003eMtING\u003c/em\u003e genes is required for some of the double mutant phenotypes. To test this, the ING domain coding sequence from \u003cem\u003eMtING1\u003c/em\u003e and \u003cem\u003eMtING2\u003c/em\u003e was cloned and fused to a recombinant N-terminal tag sequence (TRX or MBP) for expression in \u003cem\u003eE. coli\u003c/em\u003e. Purified MtING-ING domain protein (MtING\u003csub\u003eING\u003c/sub\u003e) was then tested for oligomerization \u003cem\u003ein vitro\u003c/em\u003e using SEC-MALLS analysis. When analysed alone, recombinant TRX:MtING1\u003csub\u003eING\u003c/sub\u003e or MBP:MtING2\u003csub\u003eING\u003c/sub\u003e eluted with a predicted weight-average molecular weight (MW) consistent with monomeric proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF/G). Furthermore, an equimolar mixture of both proteins eluted as two separate peaks with each peak having a MW consistent for two independent monomeric proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Thus, the ING domain of the MtING proteins does not dimerise in solution either as homo- or hetero-dimers.\u003c/p\u003e \u003cp\u003eIn summary, while both ING proteins are located in the plant cell nucleus, the SEC-MALLS analysis indicated that the MtING proteins did not interact to form either homodimers or heterodimers in solution \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene expression analyses in the\u003c/b\u003e \u003cb\u003eMting1-7 Mting2-2\u003c/b\u003e \u003cb\u003edouble knockout mutant is consistent with its poor growth and non-flowering phenotypes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the molecular basis of the strongly abnormal phenotypes of the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant, we analysed leaf and shoot apex gene expression in the double mutant, other \u003cem\u003eMting\u003c/em\u003e mutants and WT in VLD by RNA-seq and RT-qPCR (Tables S1-S5, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As expected from the strong mutant phenotypes, gene expression in the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant differed the most from WT out of all the mutant genotypes. It was strongly separated from all other genotypes in the Principle Component Analysis (PCA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) with thousands of differentially expressed genes (DEGs) compared to WT; 5189 and 3017 upregulated genes and 3064 and 1427 down regulated genes, in leaves and shoot apices respectively (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The \u003cem\u003eMting2-2\u003c/em\u003e single knockout mutant also separated distinctly from WT in the PCA analysis, and had the next highest number of DEGs; 2106 and 660 upregulated and 758 and 326 genes down regulated in leaves and shoot apices respectively compared to WT (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA/B). This is consistent with the abnormal growth and developmental phenotypes of \u003cem\u003eMting2-2\u003c/em\u003e. In contrast, the \u003cem\u003eMting1-7\u003c/em\u003e single knockout mutant clustered separately from WT in apex, but not in the leaves, and had fewer DEGs; 504 and 159 upregulated and 250 and 143 down regulated genes in leaves and shoot apices respectively compared to WT (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The remaining \u003cem\u003eMting\u003c/em\u003e single and double PHD mutants, clustered with WT in both the leaf and shoot apex samples in the PCA. This included the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD finger mutant with 584 and 140 up regulated genes and 124 and 41 downregulated genes in the leaf and shoot apex respectively. The most strongly down-regulated gene (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S4) in \u003cem\u003eMting2\u003c/em\u003e single and double mutants relative to WT, in shoot apices and/or leaves, was a candidate histone acetyltransferase gene Medtr5g017020. The predicted encoded protein is of the HAT-KAT11 superclass, in the p300/CBP HAT family (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S4). Interestingly, a second, histone acetylation related gene, Medtr5g085310 (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), was strongly down regulated in all \u003cem\u003eMting2\u003c/em\u003e single and double mutants in both tissues. This gene encodes a protein of the HAT-KAT11 superclass in the Zf-TA2 family. Amongst the most strongly up-regulated genes, we noted that in most \u003cem\u003eMting2\u003c/em\u003e mutants in leaves, these included stress-related genes encoding heat shock protein 70 (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S4) and a MYB/SANT domain gene (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knock out mutant did not produce flowers, and two other \u003cem\u003eMting\u003c/em\u003e mutants had delayed flowering, we then examined the expression of candidate Medicago flowering regulators [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] in these mutants in RNA-seq and by RT-qPCR (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S4, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC/E). In the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant many MADs genes involved in flowering were expressed at lower levels in the RNA-seq in shoot apices relative to WT. These included the inflorescence meristem identity genes \u003cem\u003eMtAP1\u003c/em\u003e and \u003cem\u003eMtAP1b\u003c/em\u003e, the I2 meristem identity gene \u003cem\u003eMtFULc\u003c/em\u003e and paralogs \u003cem\u003eMtFULa-b\u003c/em\u003e as well as \u003cem\u003eMtSEP1,4\u003c/em\u003e and \u003cem\u003eMtSOC1a-c.\u003c/em\u003e Conversely, candidate floral repressors such as \u003cem\u003eSVP-\u003c/em\u003elike genes and \u003cem\u003eMtTEM1\u003c/em\u003e and \u003cem\u003eMtTEM2\u003c/em\u003e were elevated in the mutant. Similarly, further analysis of expression of 21 candidate flowering genes by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) revealed that 15 of them were significantly differentially expressed (11 down and 4 up) in the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant relative to WT. The down regulated genes included the MADS genes above, as well as the potent floral activator \u003cem\u003eMtFTa1\u003c/em\u003e and paralog \u003cem\u003eMtFTb2\u003c/em\u003e, and \u003cem\u003eMtTFL1a\u003c/em\u003e which promotes primary inflorescence identity. Up regulated genes included the candidate floral repressors \u003cem\u003eMtBFT, MtTFL1c\u003c/em\u003e and \u003cem\u003eMtSVPc.\u003c/em\u003e RNA-seq of these genes generally followed the same pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The \u003cem\u003eMting2-2\u003c/em\u003e single knockout mutant showed strongly delayed flowering. It had reduced expression of \u003cem\u003eMtFTa1\u003c/em\u003e and the MADS floral activators and elevated expression of candidate repressors in the RNA-seq as previously reported [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD mutant had a mild delay to flowering. Amongst the MADs box genes, this correlated with down regulation of the important flowering regulator \u003cem\u003eMtAP1\u003c/em\u003e and \u003cem\u003eMtSEP4\u003c/em\u003e compared to WT. On the other hand, the \u003cem\u003eMting1-7\u003c/em\u003e single knockout mutant flowered similarly to WT and had a similar expression of candidate flowering regulators compared to WT.\u003c/p\u003e \u003cp\u003eSince the strong mutant phenotypes and abnormal gene expression of the \u003cem\u003eMting\u003c/em\u003e knockout mutants compared to WT indicated major disruption to regular biological processes, we then carried out a Gene Ontology enrichment analysis for the genes up and down regulated in the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant and other \u003cem\u003eMting\u003c/em\u003e mutants compared to WT (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). We saw that the differentially expressed genes in \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e were enriched in many biological processes. In the upregulated genes these included pathways related to plant signalling and stress such as defence and biotic stimulus responses, consistent with the upregulation of the heat shock protein 70. Down regulated enriched processes included photosynthesis, chloroplast-related and metabolism, which was consistent with the very poor growth of the mutant. In the \u003cem\u003eMting2-2\u003c/em\u003e single knockout mutant, up-regulated enriched pathways in the leaf included core processes such as rRNA processing, while in the shoot apex, upregulated genes were enriched for core processes such as transcription by RNA Polymerase III and in plant responses to biotic stimuli and defence chemical metabolism, like the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knock out mutant. Downregulated genes in \u003cem\u003eMting2-2\u003c/em\u003e in both tissues had similarities to the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant with enrichment in pathways related to photosynthesis and metabolism. The \u003cem\u003eMting1-7\u003c/em\u003e mutant had a different profile with upregulated genes in the apex showing enrichment in processes including cellular amide metabolic processes, while in leaf there was enrichment in lipid metabolic processes. There was enrichment in amine biosynthetic processes in apex down regulated genes but no enrichment amongst the down regulated genes in the leaf.\u003c/p\u003e \u003cp\u003eAlthough there have been no described mutants, AtING2 has been shown to interact with components of the NuA4 acetyltransferase complex [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, examination of the NuA4 scaffold protein Enhancer of Polycomb-Like 1 (AtEPL1) double a and b mutant (\u003cem\u003eAtepl1\u003c/em\u003e) showed that the plants were small, pale and had a downregulation in genes related to the chloroplast [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This is quite similar to the phenotype and differentially expressed genes we saw in the \u003cem\u003eMting2-2\u003c/em\u003e single knockout and \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutants (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, we examined if Medicago homologs of a list of gene targets of NuA4 in Arabidopsis were differentially expressed in the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant and respective single mutants. The Arabidopsis NuA4 targeted genes were defined previously by a loss of H4K5 acetylation and downregulation in the mutant compared to WT, as determined by ChIP-seq and RNA-seq [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, in the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant and respective single mutants, there was a marked down regulation of the selected genes in the apex tissue, with \u0026gt;\u0026thinsp;87% of the genes below a log2(\u003cem\u003eMutant\u003c/em\u003e\u003csub\u003eTPM\u003c/sub\u003e / WT\u003csub\u003eTPM\u003c/sub\u003e) of 0 (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The magnitude of the movement was also greater in the double mutant, with a median value of -0.72 compared to -0.38 and \u0026minus;\u0026thinsp;0.44 in the \u003cem\u003eMting1-7\u003c/em\u003e and \u003cem\u003eMting2-2\u003c/em\u003e single mutants, respectively. This is opposite to the general pattern of all apex DEGs, which were majority upregulated in \u003cem\u003eMting2-2\u003c/em\u003e and \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e or neutral in \u003cem\u003eMting1-7\u003c/em\u003e (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). There was a similar but weaker pattern in the leaves, with log2(\u003cem\u003eMutant\u003c/em\u003e\u003csub\u003eTPM\u003c/sub\u003e/WT\u003csub\u003eTPM\u003c/sub\u003e) median values of -0.25, -0.20 and \u0026minus;\u0026thinsp;0.33 in the \u003cem\u003eMting1-7\u003c/em\u003e, \u003cem\u003eMting2-2\u003c/em\u003e and \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e mutants respectively. Overall, this indicates that the MtINGs promote the expression of Medicago homologs of the NuA4 Arabidopsis target genes in WT. Conversely, none of the PHD finger mutants skewed in either direction (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), suggesting that the PHD finger does not contribute to the regulation of this subset of genes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe \u003cem\u003eING\u003c/em\u003e gene family is an important group of eukaryotic genes that are involved in the epigenetic regulation of a broad range of cellular processes such as gene regulation, apoptosis, senescence, cell cycle and rRNA synthesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Despite this, relatively little is known about the genetic function of the two plant \u003cem\u003eING\u003c/em\u003e genes, \u003cem\u003eING1\u003c/em\u003e and \u003cem\u003eING2\u003c/em\u003e genes, especially in regards to the regulation of flowering time which is critical for crop productivity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the\u003c/b\u003e \u003cb\u003eMting1-7 Mting2-2\u003c/b\u003e \u003cb\u003edouble knockout mutant indicates combined essential roles of the two\u003c/b\u003e \u003cb\u003eMtING\u003c/b\u003e \u003cb\u003egenes in flowering\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the combined role of the Medicago \u003cem\u003eING\u003c/em\u003e genes \u003cem\u003eMtING1\u003c/em\u003e and \u003cem\u003eMtING2\u003c/em\u003e in flowering time and growth we generated \u003cem\u003eMting\u003c/em\u003e double knockout mutants. The late flowering, short stature \u003cem\u003eMting2-2\u003c/em\u003e mutant was crossed to the WT-like \u003cem\u003eMting1-7\u003c/em\u003e mutant. The resulting \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutants were even more dwarfed, did not maintain outgrowing branches and never flowered. Previous genetic studies of other key Medicago flowering genes have shown that \u003cem\u003eMtfta1 Mtfda\u003c/em\u003e double mutants and \u003cem\u003eMtsoc1a-c\u003c/em\u003e triple mutants are also non-flowering [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Both of these mutants produced one lateral structure at the leaf axil indicative of remaining vegetative [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Because the branches of \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e mutants withered shortly after initiating, we could not determine the lateral structures in the axil. However, gene expression analysis in the shoot apex showed that key activators upstream of the floral transition including \u003cem\u003eMtFTa1\u003c/em\u003e and all three \u003cem\u003eMtSOC1\u003c/em\u003e genes, and markers of Medicago compound inflorescence meristem identity including \u003cem\u003eMtAP1, MtTFL1a\u003c/em\u003e and \u003cem\u003eMtFULc\u003c/em\u003e genes were significantly downregulated compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, the putative flowering repressors \u003cem\u003eMtSVPc\u003c/em\u003e, \u003cem\u003eMtTFL1c\u003c/em\u003e and \u003cem\u003eMtBFT\u003c/em\u003e were upregulated. In addition, \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e showed\u0026thinsp;\u0026gt;\u0026thinsp;2 times as many differentially expressed genes amongst candidate flowering gene lists than other \u003cem\u003eMting\u003c/em\u003e mutants, including the late flowering \u003cem\u003eMting2-2\u003c/em\u003e single mutant. The combined misexpression of some of these genes, together with the greatly reduced \u003cem\u003eMtTFL1a\u003c/em\u003e expression and elevated \u003cem\u003eMtSVPc\u003c/em\u003e expression compared to the \u003cem\u003eMting2-2\u003c/em\u003e single mutant, likely contributed to the double knock out mutant remaining vegetative. In summary, the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant is highly dwarfed and is unable to transition to flowering. This is overall strikingly different from the single mutant parents, \u003cem\u003eMting2-2\u003c/em\u003e with delayed flowering and small stature and \u003cem\u003eMting1-7\u003c/em\u003e which appears similar to WT, indicating combined essential roles of the two \u003cem\u003eMtING\u003c/em\u003e genes in flowering.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCombined\u003c/b\u003e \u003cb\u003eMtING\u003c/b\u003e \u003cb\u003egene effects on growth and architecture\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBeyond flowering time, the strong and diverse phenotypes of the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant including great reduction in plant height, changes to compound leaf morphology, loss of branching and large global changes to gene expression compared to WT and the single mutant parents, suggests that the two \u003cem\u003eMtING\u003c/em\u003e genes have a combined strong pleiotropic effect on gene expression and plant development. Biochemical analyses in Arabidopsis suggested that AtING2 is a scaffold component of the NuA4 acetyltransferase complex with roles in promoting chloroplast development and photosynthesis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Similarly, photosynthesis and chloroplast-related processes were enriched amongst the down regulated genes in the \u003cem\u003eMting2-2\u003c/em\u003e single mutant and in the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). There was an overall reduction in the level of expression of Medicago homologs of Arabidopsis NuA4 target genes particularly in the shoot apex in the \u003cem\u003eMting1-7\u003c/em\u003e, \u003cem\u003eMting2-2\u003c/em\u003e and \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double mutant compared to WT, with a greater magnitude of reduction in the double mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Interestingly, stress response processes were enriched in the upregulated genes in \u003cem\u003eMting2-2\u003c/em\u003e and \u003cem\u003eMting1-7 Mting2-2.\u003c/em\u003e This is also seen for NuA4 mutants such as \u003cem\u003eAtepl1\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These results are overall consistent with the idea that MtING1 and MtING2 may regulate gene transcription through histone modification such as through NuA4 action. However, Arabidopsis AtING2 can also bind to Histone Deacetylase Complex 1 (HDC1) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] a possible component of plant histone deacetylase machinery [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] indicating that MtING functions extend beyond NuA4. This is supported by the fact that there were many more up regulated DEGs than down regulated in \u003cem\u003eMting2-2\u003c/em\u003e and \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e mutants compared to WT (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This indicates that the MtINGs overall act as a repressor of gene expression rather than an activator [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the\u003c/b\u003e \u003cb\u003eMting1-1 Mting2-11\u003c/b\u003e \u003cb\u003edouble PHD mutant indicates that the PHD fingers are not essential for MtING function in flowering and development.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur previous work with \u003cem\u003eMting2\u003c/em\u003e PHD finger single mutants indicated that unexpectedly the conserved PHD finger was not essential for its function, because these single mutants grew and flowered similarly to WT [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To test if the loss of the MtING2 PHD finger was being compensated for by MtING1, we made the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e PHD double mutant. Apart from a strong loss of trichomes, the double mutant showed only a mild delay to flowering time and weak effects on development and gene expression. Therefore, the PHD fingers of both MtINGs do not appear to contribute strongly to the biological function of the MtING proteins in Medicago.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur work indicates that \u003cem\u003eMtING1\u003c/em\u003e and \u003cem\u003eMtING2\u003c/em\u003e have overlapping and complementary functions with a combined essential role in the transition to flowering in floral inductive VLD conditions and in growth and development. However, interestingly despite their conservation, the PHD fingers of the MtINGs are not critical for MtING function in flowering and growth. This implicates the other conserved domain, the N-terminal ING domain, as perhaps the most important element for MtING function. Future work should investigate whether the MtINGs are components of chromatin modifying complexes such as a Medicago NuA4 acetyltransferase complex and what genes might be direct targets of this complex. More broadly, based on the foundations provided by our work, it will be important to investigate the genetic functions of the \u003cem\u003eINGs\u003c/em\u003e in growth, development and flowering in other plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eEthics approval and consent to participate.\u0026nbsp;\u003c/strong\u003e\u0026ldquo;Not applicable\u0026rdquo;.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e \u0026ldquo;Not applicable\u0026rdquo;.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u0026ldquo;Not applicable\u0026rdquo;.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAvailability of data and materials.\u003c/strong\u003e The datasets generated and/or analysed during the current study are available in the GEO repository (GSE277907).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003e\u0026ldquo;Not applicable\u0026rdquo;.\u0026nbsp;The authors declare that they have no competing interests.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThis work was funded by the Royal Society of New Zealand Te Apārangi through a Marsden grant awarded to J.P (contract 17-UOA-075, used to support M.M-S).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAuthors\u0026apos; contributions.\u0026nbsp;\u003c/strong\u003eMMS generated the \u003cem\u003eMting\u003c/em\u003e double mutants, phenotyped the plants, carried out the nuclear localization and SEC-MALLS with DG. LZ performed plant crossing, RNA isolation and RT-qPCR. AP processed and analysed RNA-seq data and YP curated RNA-seq data and carried out GO analysis. JP and DG conceptualized the project, wrote the original draft with MMS and reviewed and edited the manuscript. All authors contributed text, and read and approved the final manuscript.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAcknowledgements.\u0026nbsp;\u003c/strong\u003eWe would like to acknowledge Nicky Vernon, Nathan Deed, Adrian Turner and the members of the Goldstone laboratory for their technical support.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGraham PH, Vance CP. Legumes: importance and constraints to greater use. \u003cem\u003ePlant physiology \u003c/em\u003e2003; 131(3):872-877. \u003c/li\u003e\n\u003cli\u003eClarkson N, Russell J. 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Histone deacetylase complex1 expression level titrates plant growth and abscisic acid sensitivity in Arabidopsis. \u003cem\u003eThe Plant Cell \u003c/em\u003e2013; 25(9):3491-3505. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"INHIBITOR OF GROWTH, MtING1, MtING2, legume, Medicago, Arabidopsis, flowering time, PHD finger, ING domain","lastPublishedDoi":"10.21203/rs.3.rs-5314612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5314612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOptimal flowering time is critical to agricultural productivity. Despite this, the molecular mechanisms regulating flowering in the economically important Fabaceae (legume) family are not fully understood. For example, the key flowering regulators known from Arabidopsis, FLC and CO, do not regulate flowering in the temperate model legume \u003cem\u003eMedicago truncatula\u003c/em\u003e (Medicago). Previously, we used CRISPR-Cas9 mutagenesis to show the histone modification reader MtINHIBITOR OF GROWTH 2 promotes flowering and growth in Medicago. However, surprisingly, the highly conserved C-terminal plant homeodomain (PHD) finger did not appear to contribute to this, as \u003cem\u003eMting2\u003c/em\u003e PHD finger mutants flowered and grew similarly to wild type. Additionally, a second \u003cem\u003eING\u003c/em\u003e gene, \u003cem\u003eMtING1\u003c/em\u003e, did not appear to regulate flowering.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo further dissect the genetic function of the two \u003cem\u003eMtING\u003c/em\u003e genes in flowering and growth, we cross-pollinated selected \u003cem\u003eMting1\u003c/em\u003e and \u003cem\u003eMting2\u003c/em\u003e single mutants to create two different double mutants; the \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutant and the \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD finger mutant. The growth and flowering of these mutants was assessed in floral-inductive vernalised long day conditions. We also used fluorescence confocal microscopy and \u003cem\u003ein vitro\u003c/em\u003e protein biophysical analysis to investigate the subcellular localization and oligomerization of the proteins. Finally, we carried out gene expression analysis by RNA-seq and RT-qPCR to determine how the two genes affect transcript accumulation to influence growth and flowering.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eMting1-7 Mting2-2\u003c/em\u003e double knockout mutants were very small, did not maintain outgrowing branches and never flowered. \u003cem\u003eMting1-1 Mting2-11\u003c/em\u003e double PHD finger mutants on the other hand showed only mild dwarfing and delays to flowering. GFP tagged MtING proteins localised to the nucleus in tobacco leaves. However, recombinant MtING domain proteins did not form dimers in solution. Gene expression analyses showed large changes to global gene expression in the double knockout mutant with key flowering genes downregulated and predicted floral repressors elevated.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAlthough the first \u003cem\u003eING\u003c/em\u003e gene was identified in humans almost three decades ago, very little is known about the two plant \u003cem\u003eING\u003c/em\u003e genes. Our findings demonstrate the essential combined role the \u003cem\u003eMtING\u003c/em\u003e genes play in the regulation of gene expression, flowering time and wider development.\u003c/p\u003e","manuscriptTitle":"Medicago Mting1 Mting2 double knockout mutants are extremely dwarfed and never flower implicating essential MtING functions in growth and flowering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-07 08:09:48","doi":"10.21203/rs.3.rs-5314612/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-05T10:35:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-25T04:59:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-25T04:58:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-10-22T23:51:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c61ab3d-b672-45bf-a4b6-f5f9ebb64867","owner":[],"postedDate":"November 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T16:06:38+00:00","versionOfRecord":{"articleIdentity":"rs-5314612","link":"https://doi.org/10.1186/s12870-025-06432-x","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-04-01 15:57:50","publishedOnDateReadable":"April 1st, 2025"},"versionCreatedAt":"2024-11-07 08:09:48","video":"","vorDoi":"10.1186/s12870-025-06432-x","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06432-x","workflowStages":[]},"version":"v1","identity":"rs-5314612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5314612","identity":"rs-5314612","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}