Functional Characterization Of MfGATA25 And MfIDD1 From Medicago Falcata And MtIAA15 From Medicago Truncatula In Regulation On Cold Tolerance And Growth

Low temperature is one of the major abiotic stresses that leads to decrease in the yield of alfalfa(Medicago sativa L.),an important forage legume.M.falcata(Medicago sativa subs.falcata L.)is a Medicago species that exhibits better cold tolerance than alfalfa and M.truncatula.M.truncatula is a model legume plant which has been similar genetic background with alfalfa and M.falcata.Based on the analysis of differentially expressed genes in response to cold between M.falcata and M.truncatula,three transcription factors,Mf GATA25,Mf IDD1 and Mt IAA15 were selected and studied in cold tolerance and growth regulation.1.Functional analysis of Mf GATA25A GATA transcription factor(Mf GATA25)was cloned from M.falacta.Mf GATA25,belongs to short B-Class GATAs family,localizes to the nucleus and acts as transcriptional repressor.Mf GATA25 transcript was induced by cold,GA and ABA treatment.Constitutive overexpression of Mf GATA25 in stylo(Stylosanthes guianensias SW.)caused major developmental defects associated with decrease height and biomass and increase in branch.Morphological observation indicated that Mf GATA25 controlled plant development via regulation of cell expansion.Compared to the WT plants,transgenic stylo plants had higher level of survival rate and maximum photochemical efficiency(Fv/Fm)after 4 days of chilling stress at 4?.In addition,an elevated activity of antioxidant enzymes was observed in transgenic stylo plants compared to WT plants.RNA-seq analysis revealed that genes involved in auxin and gibberellin signaling were down-regulated,while the content of IAAs and GAs showed no difference between transgenic stylo and WT plants.There were 249differentially expressed genes in transgenic stylo and WT plants under chilling treatment,which focused on signal transduction,material synthesis and DNA repair.The expression of flavonoids-related genes in transgenic stylo was significantly up-regulated.Therefore,it is suggested that Mf GATA25 reduces height and biomass of stylo by negatively regulates the length and size of cells and improves cold resistance.2.Functional analysis of Mf IDD1IDD family(Indeterminate Domain)is specifically existed in plants,however,its function is largely unknown.Mf IDD1 contains four zinc finger domain,two C2H2 zinc finger domain and two C2HC zinc finger domain,and two conserved sequence at C terminus.Mf IDD1 localized to the nucleus and acts as transcriptional activator.Mf IDD1 expression was induced by gibberellin and cold,but not altered in response to auxin and ABA treatment.Transgenic M.truncatula overexpressing Mf IDD1 showed phenotype with delayed seed germination and flowering and dwarfism.Furthermore,seed germination in transgenic M.truncatula plants was hypersensitive to GA and ABA.In addition,transgenic plants showed no difference in cold tolerance compared with the wild the wild type based on the data of LT₅₀(temperature in 50%lethal)and Fv/Fm.Our results indicated that Mf IDD1 controls germination,development and flowering.3.Functional analysis of Mt IAA15The role of Mt IAA15(Auxin/Indole acetic acid 15)from M.truncatula in cold tolerance was studied.Mt IAA15 was confirmed to be localized at the nucleus and cytoplasm,may act as a transcriptional repressor.Mt IAA15 transcript was induced by cold and auxin.Degradation of Mt IAA15-GFP induced by auxin,while Mt IAA15^P114S protein become resistant to auxin when conserve amino acids“GWPPV”in domain?was altered by mutation.Mt IAA15 was expressed in hypocotyl after germination.Then,Mt IAA15 was detected in young leaves,branch,root tip and rhizobia except in mature and old leaves during vegetative stage.The expression of Mt IAA15 was existed in flower and capsule at the reproductive stage.Transgenic plant analysis overexpressing Mt IAA15 showed an elevated freezing tolerance as compared with the wild type,with unaltered growth and development phenotypes.By using yeast hybridization and Bi FC(bimolecular fluorescence complementation),six candidate interaction protein with Mt IAA15 were identified.Among them,Mt ARF23(auxin response factor 23)and Mt ARF25 were confirmed to be located at the nucleus with transcriptional activator and transcriptional repressor respectively.In conclusion,Mf GATA25,which was induced by low temperature in M.falcata,inhibits plant growth and improves cold resistance.Mf IDD1 gene expression is less affected by low temperature and mainly control seed germination,plant growth and flowering.Mt IAA15,its transcript was induced by low temperature,could be regulate cold tolerance by interacting with Mt ARF23 and Mt ARF25.