Genetic Effects Of GmFT2a And GmFT5a On Floral Induction In Soybean

Soybean(Glycine max(L.)Merr.)is known as a short-day plant that accelerates transition from the vegetative phase to the reproductive phase when it senses short-day(SD)conditions.Soybean still flowers under long-day(LD)conditions,albeit much later than in SD conditions.Flowering time is a key agronomic trait that directly influences the successful adaptation of soybean to diverse latitudes and farming systems.Floral transition is synchronized by the integration of signals from factors that are both endogenous and environmental.Photoperiod is one of the major determinants of soybean's adaptation to seasonal changes in day length for flowering.In Arabidopsis thaliana,FLOWER LOCUS T(FT)plays an important role in flowering pathways as an integrator and encodes a florigen protein that functions as a long-distance signal to induce floral initiation.Ten FT homologs have been identified in soybean.Among them,GmFT2a(Glyma.16G150700)and GmFT5a(Glyma.16G044100)have been confirmed to have main promoting effects on flowering time.The purpose of this study was to dissect individual effects of GmFT2 a and GmFT5 a under SD and LD conditions.The coding regions of GmFT2 a and GmFT5 a are conserved,and there are fewer natural ft2 a or ft5 a mutants.In this study,we created ft2 a mutants and ft5 a mutants by CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9)with mutation efficiencies of36.7%-52.8% and 70.8%-73.7%,respectively.Frameshift mutations induced by CRISPR/Cas9 at target sites of GmFT2 a or GmFT5 a generated premature translation termination codons(PTCs),and the mRNA of GmFT2 a or GmFT5 a in the corresponding mutants showed lower expression levels than that of WT plants.We identified ‘transgene-free' soybean plants that were homozygous for null alleles of endogenous GmFT2 a or GmFT5 a and without any transgenic element from the T1 and T2 generations.We subsequently created ft2 a ft5a double mutants by hybridization using ft2 a mutants and ft5 a mutants.In addition,we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2 a and GmFT5 a using a dual-sgRNA/Cas9 design.We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2 a.We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2 a and 15.8%for target fragments ranging from1069 to 1161 bp in GmFT5 a.We demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited.The T2 ‘transgene-free' homozygous ft2 a mutants with a 1618 bp deletion exhibited the late-flowering phenotype under SD and LD conditions.In this study,we also created transgenic plants overexpressing GmFT2 a or GmFT5a(GmFT2a-ox plants,GmFT5a-ox plants).Under SD conditions and compared to wild-type(WT)plants,the GmFT2a-ox plants displayed early flowering by about 9.4 days and the ft2 a mutants exhibited late flowering by about 5.9 days.The flowering time of GmFT5a-ox plants and ft5 a mutants were almost the same as WT plants.The ft2 a ft5a double mutants flowered at 57.4±3.5 DAE,about 31.3 days later than WT plants.Under LD conditions and compared to WT plants,the GmFT2a-ox plants showed early flowering phenotypes of about 31.7 days,and the ft2 a mutants also exhibited late flowering by about3.6 days.Unlike the observation of no significant phenotypic alterations in flowering time that we observed under SD conditions,we found that overexpression of GmFT5 a caused early flowering by about 16.2 days under LD conditions.Even more remarkably,the ft5 a mutants flowered at 124.7±15.4DAE,about 76.2 days later than WT plants.The ft2 a ft5a double mutants did not flower up to 150 DAE.Combining the results of phenotypic analysis under different environments,and expression of flowering-related genes,we study suggests that GmFT2 a and GmFT5 a collectively regulate flowering time in soybean but indicates that the effect of GmFT2 a is more prominent under SD conditions whereas the effect of GmFT5 a is more prominent under LD conditions.The ft2 a ft5a double mutants showed late flowering by about 31.3 days under SD conditions.Most nodes of the ft2 a ft5a double mutants first produced many new branches instead of flowers at the leaf axil,then produced flower buds at these new branches.Importantly,the branches at the leaf axils of the ft2 a ft5a double mutants produced more pods than the WT plants,thereby increasing the number of pods at each node.Along with the increased number of nodes(20.0±0.9 for ft2 a ft5a double mutants vs.10.3±0.7 for WT),the total numbers of pods and seeds of per ft2 a ft5a double mutant(Pods 54.7±12.6;Seeds 106.3±22.8)were significantly more than that of WT plants(Pods 11.9±3.2;Seeds 27.7±6.4).