The Study On The Mechanism Of Nyctinastic Leaf Movement In Medicago Truncatula

Many plant species open their leaves during the daytime and close them at night as if sleeping.The leaf movement,known as nyctinasty,is widely observed in leguminous and oxalidaceae plants,which are generated by a specialized motor organ,pulvinus.The PETIOLULE-LIKE PULVINUS(PLP)gene was identified as the determinant of pulvinus in previous report.In this study,we also identified the MtSAP,MtFIL and MtYAB3 play roles in the development of M.truncatula pulvinus.In addition,the brassinosteroid(BR)homeostasis is critical for development of pulvinus and nyctinastic leaf movement of M.truncatula.As sessile organisms,plants perceive,respond,and adapt to the environmental changes for optimal growth and survival.The plant growth and fitness are enhanced by circadian clocks through coordination of numerous biological events.In legume species,nitrogen-fixing root nodules were developed as the plant organs specialized for symbiotic transfer of nitrogen between microsymbiont and host,which is important for environmental improvement.So far,there is still no report about the interaction between the circadian clock and the nodules in legumes.1.Brassinosteroid Homeostasis Is Critical for the Development and Functionality of Medicago truncatula Pulvinus,But Not for Its DeterminationNyctinastic leaf movement is generated by pulvinus.Although a key determinant of pulvinus,PETIOLULE-LIKE PULVINUS(PLP)has been identified,the molecular genetic basis for the pulvinus function is largely unknown.Here,an analysis of knockout mutants in Medicago truncatula showed that neither altering the brassinosteroid(BR)content nor blocking BR signal perception would affect the pulvinus,while both did influence nyctinastic leaf movement.BR activity in the pulvinus is regulated by the product of a BR inactivating gene PHYB ACTIVATION TAGGED SUPPRESSOR1(BAS1),which is directly activated by PLP.A comparative analysis between M.truncatula and the non-pulvinus forming species Arabidopsis and tomato revealed that the product of the gene PLP has become a determinant of pulvinus formation in a species-specific manner.Apart from exposing the involvement of BR in the development of the pulvinus,the experiments have provided insights into the general or specialized gene functions among species.2.Regulation of Medicago truncatula pulvinus development by PETIOLULE-LIKE PULVINUS(PLP)In this study,we identified mtsap mutant with defective leaf movement by forward genetic screening.Overexpression of PLP in mtsap mutant is able to recover the defects of pulvinus,indicating that PLP is genetically downstream of MtSAP.However,ectopic expression of MtSAP cannot upregulate the expression level of PLP,demonstrating that MtSAP plays positive role in the pulvinus development by regulating PLP indirectly.Besides,two homologous YABBY genes of Arabidopsis,MtFIL and MtYAB3,are involved in the formation of pulvinus.In the mtfil mtyab3 double mutants,the pulvinus is changed to petiolule-like structure,which is similar with plp.PLP functions in activating the transcription of MtFIL and MtYAB3.Meanwhile the yeast two-hybrid and BiFC assay showed the physical interaction between PLP and MtFIL/MtYAB3 in M.truncatula,as well as those in other legumes.Therefore,we made a hypothesis that this complex regulates multiple signaling pathways that promote the initiation and maintenance of pulvinus in legume.This result will shed insights into the roles of polarity related genes in controlling of pulvinus development.3.The nodulation and nyctinastic leaf movement is orchestrated by clock gene LHY in Medicago truncatulaAs sessile organisms,plants perceive,respond,and adapt to the environmental changes for optimal growth and survival.The plant growth and fitness are enhanced by circadian clocks through coordination of numerous biological events.In legume species,nitrogen-fixing root nodules were developed as the plant organs specialized for symbiotic transfer of nitrogen between microsymbiont and host.Here,we report that the endogenous circadian rhythm in nodules is regulated by MtLHY in legume species Medicago truncatula.Loss-of-function of MtLHY leads to a reduction in the number of nodules formed,resulting in a diminished ability to assimilate nitrogen.The operation of the 24-h rhythm in shoot is further influenced by the availability of nitrogen produced by the nodules,leading to the irregulated nyctinastic leaf movement and reduced biomass in mtlhy mutants.These data shed new light on the roles of MtLHY in the orchestration of circadian oscillator in nodules and shoots,which provides a mechanistic link between nodulation,nitrogen assimilation and clock function.