Functional Analysis Of MtKNOX4 And MtUFO In Compound Leaf Development In Medicago Truncatula

Leguminous plants are widely distributed,including 650 genera and 18000 species.Currently Medicago trucatula is being used as a model plant of legume since it has a modest diploid genome,self-fertile nature,compound leaf developmental speciality,which are particularly important for scientific research.We isolated a leaf-developmental mutant,mtknox4 through forward genetic screening and cloned its full length CDS from wild type R108.The CDS of MtKNOX4 is 882bp and encodes 293 amino acids,which belong to Class ?KNOTTED-LLKE HOMEBOX gene family.The adult leaves of mtknox4 mutant exhibited four or five leaflets instead of the typical ternately compound leaf of wild type.The length of petiole and rachis became significantly shorter,but the leaflet number was unchanged when MtKNOX4 was overexpressed.Real time quantative PCR(qRT-PCR)was used to analyse the expression level of GA and CK pathways related genes in mtknox4 mutant and MtKNOX4 overexpression plants.We found that the expression level of almost all genes showed no obvious changes.However,we found that 7 candidate genes in MtBELL family may form a dimer with MtKNOX4 by yeast two-hybrid system.All these results imply that the variation of leaflets number in mtknox4 may be caused by interaction with MtBELL.But the exact molecular mechanisms are still unknown.In addition,we obtained another gene related to floral organs development,UNUSUAL FLORAL ORGANS,of which overexpression lines would present the increased leaflets number.qRT-PCR data showed no uniform changes in GA signal pathway related genes in overexpression MtUFO lines,compared with those in wild type.Neither exogenous active GA3 application nor GA2oxl(Medtr8g461330)overexpression could cause the change of leaflets number,implying that GA may be not related with the formation of ectopic leaflets.In previous study,loss-of-function in SGL1 caused simple adult leaves due to failure in initiating lateral leaflet primordia,which are the same with 35S-MtUFO sgl1 double combination.These results suggested the formation of ectopic leaflets in overexpression MtUFO plant was dependent on the function of SGL1.Yeast two-hybrid results showed SGL1 had no interaction with MtUFO,while its homologous genes did in Arabiddopsis thaliana.No interaction between the two proteins may be due to the deletion of several functional amino acids in the C-terminal site of protein SGL1.