Conserved functions of angiosperm ε-clade 14-3-3 proteins in growth, flowering, and abiotic stress resilience

14-3-3 proteins are involved in a number of developmental processes in plants, largely acting through the control of transcription factor localization within the cytoplasm or nucleus. The temperate Pooideae grass 14-3-3 gene GF14h in particular was previously found to be co-expressed with flowering activators in the vernalization pathway, leading to the hypotheses that its protein product is involved in vernalization responsive flowering and/or cold acclimation. To test predictions of these non-exclusive hypotheses, and to assess functional conservation broadly across angiosperms, we generated CRISPR lines of GF14h in Brachypodium distachyon (Pooideae) and compared their mutant phenotypes with those of the co-orthologous Arabidopsis thaliana (Brassicaceae) grf9 T-DNA mutant. Contrary to our first prediction, we found that rather than promoting flowering as part of the vernalization pathway, both BdGF14h and AtGRF9 repress flowering under different conditions. Our data also support a conserved role for GF14h/GRF9 genes in the promotion of growth at the expense of abiotic stress resilience. We discuss these results in the light of crop improvement and climate change. Total RNA was extracted from whole aboveground tissues of plants at the five leaf stage at the end of a 15 day drought treatment. Drought was conducted by withholding water. All material was collected three hours post dawn to control for circadian rhythms in gene expression. RNAseq was conducted for cDNA libraries on three biological replicates of wild type Brachypodium distachyon 21-3 and mutant (CR:GF14h-1 and CR:HDAC19-2b) plants of the same genotypic background as the wild type. Mutations were specific to the 14-3-3 protein encoding gene GF14h or HISTONE DEACETYLASE 19 (HDAC19) based on CRISPR/Cas9 editing and subsequent genotyping. Mutants for both genes are homozygous for a mutation in exon 1 and the mutations cause a premature stop codon.