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

Background: Optimal flowering time is critical to agricultural productivity. Despite this, flowering regulation in the Fabaceae (legume) family is not fully understood. For example, FLC and CO control Arabidopsis flowering, but do not regulate flowering in the temperate legume Medicago. Little is known about the genetic roles of the two plant ING genes. They encode proteins with conserved ING and PHD finger domains predicted to function as epigenetic readers. Previously, using CRISPR-Cas9 knock outs, we reported that Medicago MtING2 promotes flowering and growth. However, surprisingly, Mting2 PHD finger mutants flowered similarly to wild type. Additionally, MtING1 did not regulate flowering because Mting1 mutants flowered like wild type.

Methods: To further dissect the combined genetic function of MtING1 and MtING2 and their PHD fingers, we cross-pollinated Mting1 and Mting2 single mutants to create two double mutants: The Mting1-7 Mting2-2 double knockout mutant and the Mting1-1 Mting2-11 double PHD finger mutant. Mutant phenotypes were assessed in floral-inductive conditions. We used fluorescence confocal microscopy and in vitro protein biophysical analysis to investigate the subcellular localization and oligomerization of the proteins. 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 Mting double knockout mutants displayed a striking, non-flowering, highly dwarfed phenotype indicating overlapping and complementary functions. Conversely Mting double PHD finger mutants showed only mild dwarfing and weak delays to flowering, indicating that the PHD fingers did not have a major impact on MtING function. MtING proteins localised to the nucleus, consistent with their predicted roles as histone readers, but did not interact in solution. Large changes to gene expression were seen in the Mting2-2 single mutant and the double knockout mutant, with key flowering genes downregulated and predicted floral repressors elevated. Furthermore, the MtINGs promoted the expression of Medicago homologs of target genes of the Arabidopsis NuA4 HAT complex.

Conclusions: Our findings demonstrate the key combined function the MtING genes play in regulation of global gene expression, flowering time and wider development and implicate an important role in epigenetic regulation via HAT complexes.