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

The regulation of flowering time to synchronise with external environmental signals is critical to plant reproductive success and agricultural productivity. Previously, we used CRISPR-Cas9 gene editing to show the INHIBITOR OF GROWTH 2 gene (MtING2) promotes flowering and growth in the model temperate legume Medicago truncatula, especially under inductive vernalized long days. A second ING gene, MtING1, did not appear to regulate flowering. To further dissect the genetic function of the two ING genes in flowering and growth, we cross-pollinated selected Mting1 and Mting2 single mutants to create two different double mutants; Mting1-7 Mting2-2 double knockout mutant and Mting1-1 Mting2-11 double PHD finger mutant. The growth and flowering of these mutants was assessed in vernalized 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 both RNA-seq and RT-qPCR to determine how the two genes affect transcript accumulation to influence flowering. Overall design: We carried out RNA-seq on single Mting mutants and double Mting mutant and compared to the relative wildtype. 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 plants had 2-3 fully expanded trifoliate leaves. Three biological replicates were harvested per tissue type per genotype, each consisting of 1 to 2 fully expanded trifoliate 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, Germany) according to the user manual. RNA quantity and quality were checked by a NanoPhotometer® N60 (Implen, Germany) and a Bioanalyzer 2100 (Agilent Technologies, USA).