<scp><i>MtING2</i></scp> encodes an <scp>ING</scp> domain <scp>PHD</scp> finger protein which affects Medicago growth, flowering, global patterns of <scp>H3K4me3</scp>, and gene expression

Jaudal, Mauren; Mayo-Smith, Matthew; Poulet, Axel; Whibley, Annabel; Peng, Yongyan; Zhang, Lulu; Thomson, Geoffrey; Trimborn, Laura; Jacob, Yannick; Wolfswinkel, Josien C. van; Goldstone, David C.; Wen, Jiangqi; Mysore, Kirankumar S.; Putterill, Joanna

doi:10.1111/tpj.15994

Cited by 5 publications

(8 citation statements)

References 117 publications

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“…MtFDa was also downregulated at the apex of the triple mutant. The candidate floral repressors MtFTa2 and MtSVPc were upregulated in the triple mutant ( Figure 4D ), consistent with their elevation in the late-flowering Mting2 mutant ( Jaudal et al., 2022 ), indicating that they are likely to play a repressive role in Medicago flowering. MtFULb and MtSOC1c levels were elevated in the triple mutant, suggesting their misregulation in the shoot apex.…”

Section: Discussionsupporting

confidence: 59%

“…The flowering time and I2 inflorescence meristem identity gene, MtFDa , were also downregulated. Four genes, including the likely flowering repressor MtSVPc and candidate FT-like flowering repressor MtFTa2 ( Jaudal et al., 2022 ), were upregulated in the triple mutant. RNA-seq analysis of the triple Mtsoc1 mutant yielded similar results.…”

Section: Resultsmentioning

confidence: 99%

“…One construct consisting of a polycistronic pre-tRNA-sgRNA-scaffold cassette ( Xie et al., 2015 ) placed downstream of the Medicago U6 promoter with all seven guides and HindIII restriction sites added on both ends was commercially synthesized by GenScript (USA). The construct was inserted into the pCBSG041 plasmid vector backbone with Cas9 driven by the constitutive CAMV 35S promoter, as previously described ( Jaudal et al., 2022 ). The plasmid was transformed into Agrobacterium tumefaciens strain EHA105 to transform Medicago WT R108 leaf tissue.…”

Section: Methodsmentioning

confidence: 99%

“…The polycomb repressive complex 2 component MtVERNALISATION2 represses Medicago flowering until after vernalization ( Jaudal et al., 2016 ). MtINHIBITOR OF GROWTH 2 ( MtING2 ) promoted flowering particularly in response to VLD ( Jaudal et al., 2022 ). The floral integrator genes include the FLOWERING LOCUS T (FT)-like gene MtFTa1 ( Laurie et al., 2011 ; Yeoh et al., 2013 ; Jaudal et al., 2020a ; Zhang et al., 2021b ) and the FLOWERING LOCUS D (FD)-like gene MtFDa.…”

Section: Introductionmentioning

confidence: 99%

“…This is unlike Arabidopsis, where the ft-10 twin sister of ft (tsf-1) fd-3 triple mutants showed only delayed flowering ( Romera-Branchat et al., 2020 ). Other likely players include multiple paralogs of genes encoding MADS transcription factors, such as the three MtSUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) ( MtSOC1a , b , c ), three MtFUL genes ( MtFULa, b, c ), three MtSHORT VEGETATIVE PHASE ( MtSVP1, 2, c ), and additional FT / TERMINAL FLOWER 1 (TFL1) genes including MtFTa2 , MtFTb1 , MtFTb2 , and MtTFL1a, c ( Laurie et al., 2011 ; Jaudal et al., 2014 , 2015 ; Sussmilch et al., 2017 ; Cheng et al., 2018 ; Fudge et al., 2018 ; Jaudal et al., 2018 ; Thomson et al., 2019 ; Jaudal et al., 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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Gene-edited Mtsoc1 triple mutant Medicago plants do not flower

Poulet,

Zhao,

Peng

et al. 2024

Front. Plant Sci.

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Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. SOC1-like genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model Arabidopsis thaliana (Arabidopsis), where SOC1 promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. Medicago truncatula (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one SOC1 gene, multiple gene duplication events have given rise to three MtSOC1 paralogs within the Medicago genus in legumes: one Fabaceae group A SOC1 gene, MtSOC1a, and two tandemly repeated Fabaceae group B SOC1 genes, MtSOC1b and MtSOC1c. Previously, we showed that MtSOC1a has unique functions in floral promotion in Medicago. The Mtsoc1a Tnt1 retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, Mtsoc1b Tnt1 single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of Mtsoc1a Mtsoc1b Mtsoc1c triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three MtSOC1 genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single Mtsoc1a mutant and the Arabidopsis soc1 mutant; implicating multiple MtSOC1 genes in critical overlapping roles in the transition to flowering in Medicago.

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Section: Discussionsupporting

confidence: 59%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Gene-edited Mtsoc1 triple mutant Medicago plants do not flower

Poulet,

Zhao,

Peng

et al. 2024

Front. Plant Sci.

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

Mayo-Smith,

Poulet,

Zhang

et al. 2024

Preprint

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Background Optimal 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 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.

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Recalcitrance to transformation, a hindrance for genome editing of legumes

Nivya,

Shah

2023

Front. Genome Ed.

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Plant genome editing, a recently discovered method for targeted mutagenesis, has emerged as a promising tool for crop improvement and gene function research. Many genome-edited plants, such as rice, wheat, and tomato, have emerged over the last decade. As the preliminary steps in the procedure for genome editing involve genetic transformation, amenability to genome editing depends on the efficiency of genetic engineering. Hence, there are numerous reports on the aforementioned crops because they are transformed with relative ease. Legume crops are rich in protein and, thus, are a favored source of plant proteins for the human diet in most countries. However, legume cultivation often succumbs to various biotic/abiotic threats, thereby leading to high yield loss. Furthermore, certain legumes like peanuts possess allergens, and these need to be eliminated as these deprive many people from gaining the benefits of such crops. Further genetic variations are limited in certain legumes. Genome editing has the potential to offer solutions to not only combat biotic/abiotic stress but also generate desirable knock-outs and genetic variants. However, excluding soybean, alfalfa, and Lotus japonicus, reports obtained on genome editing of other legume crops are less. This is because, excluding the aforementioned three legume crops, the transformation efficiency of most legumes is found to be very low. Obtaining a higher number of genome-edited events is desirable as it offers the option to genotypically/phenotypically select the best candidate, without the baggage of off-target mutations. Eliminating the barriers to genetic engineering would directly help in increasing genome-editing rates. Thus, this review aims to compare various legumes for their transformation, editing, and regeneration efficiencies and discusses various solutions available for increasing transformation and genome-editing rates in legumes.

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MtING2 encodes an ING domain PHD finger protein which affects Medicago growth, flowering, global patterns of H3K4me3, and gene expression

Cited by 5 publications

References 117 publications

Gene-edited Mtsoc1 triple mutant Medicago plants do not flower

Gene-edited Mtsoc1 triple mutant Medicago plants do not flower

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

Recalcitrance to transformation, a hindrance for genome editing of legumes

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