Role And Molecular Basis Of FT4 In Flowering Control In Pooideae Grasses

Flowering is an important process in plants to complete the reproduction of offspring,Whether plants can blossom at the appropriate time is related to the length of its entire life cycle.Several floral pathways were integrated in regulation of FLOWERING LOCUS T(FT),a florigen protein in plants.In this study,we characterized the functions of FT4 in flowering control in B.distachyon and wheat,and illustrated the molecular mechanisms that how FT4 represses heading under specific conditions.The major findings are presented as below:1.FT4 expression is photoperiod-independent.There are two FT orthologs in Arabidopsis,while six of those in B.distachyon,namely FT1 to FT6,respectively.we found that the expression of FT4 was not relied on photoperiod.these results imply a special role of FT4 in flowering regulation in B.distachyon.2.FT4 represses flowering.Overexpressed FT4 showed a delayed flowering phenotype in B.distachyon.Moreover,we found that FT4 interacted with transcription factor FD and scaffolding proteins 14-3-3 to constitute a florigen repression complex(FRC),instead of a general florigen activation complex(FAC)to inhibit flowering onset.3.Y122 as the Core amino acid required for FT4 activityIn vitro transient expression assays revealed that tyrosine(Tyr,Y)at 122 residue of FT4 was mutated to phenylalanine,which makes FT4 changed into promoted FT.These results indicated that Y122 residue was the core amino acid for the floral inhibitive activity of FT4.4.FT4 plays opposite roles with its paralog in flowering controlWe showed that ft4,a mutant generated by using CRISPR/Cas9 technique,had no significant early flowering phenotype.We then produced ft4ft6 double mutants in which FT6,the closest homolog of FT4,was deleted in B.distachyon,and found that the double mutants delayed flowering.Additionally,genetical and biochemical experiments demonstrated that FT6 was a promoter and FT4 was inhibitor of flowering control in B.distachyon.5.FT4 is degraded under normal conditions in B.distachyon.In cell-free assay,we revealed that FT4 protein was degraded depending on proteasome and ubiquitin/26 S system.Moreover,further experiments demonstrated that FT4 was degraded by ubiquitin/26 S proteasome in transgenic plants.Therefore,the instability of FT4 protein is the major cause that ft4 mutant shows a normal flowering.6.FT4 is accumulated and inhibits flowering under high nitrogen conditions.We observed that the heading of flowering in ft4 mutant was much earlier than that of WT under high nitrogen(HN)than normal nitrogen(NN)conditions.Furthermore,we determined an enhancement of both gene expression and protein of FT4 under HN conditions postponed flowering.7.The mechanism of FT4 in flowering initiation is conserved in wheat and B.distachyon.We test stability of FT4 protein encoded from B,sub-genome of tetraploid wheat,and found that FT4 protein was more stabilized under HN than NN conditions.These data implicate that the role and molecular machinery of FT4 in flowering regulation are conserved in wheat and B.distachyon.Taken together,our findings illustrate that growth transition affected by N availability is mediated by an anti-florigen protein in temperate grasses.These results may provide insights into improvement of plant fitness as well as increase of nitrogen use efficiency in wheat crops.