Arabidopsis EMS1-TPD1 Signal Regulates The Formation Mechanism Of Anther Tapetum

In flowering plants,successful sexual reproduction depends on the specification of distinct somatic and reproductive cell types that yield male gametophytes.The anther,where male gametophytes(pollen)are produced,typically has four lobes.Within each of these lobes in a mature anther,the central reproductive microsporocytes(or pollen mother cells)are surrounded by four concentrically organized somatic cell layers.Microsporocytes give rise to pollen via meiosis,while somatic cells,particularly the tapetum,are required for the normal development.Abnormalities in the tapetum can lead to reduced male fertility or infertility.Previous studies have shown that a secreted peptide TPDl,its receptor EMS1 and co-receptor SERK1/SERK2 together determine the formation of tapetum.The current model suggests that the TPD1 is secreted from the precursors of microsporocytes,which then interacts with EMS1 at the plasma membrane of the tapetum precursor cells,thereby activating the intracellular signaling pathway and determining the tapetum formation.However,little is known about the intracellular transduction pathway of EMS1-TPD1 signaling.This study has uncovered that EMS1 binds to the ligand TPD1 through an extracellular domain different from that of BRI1,activates the conserved intracellular domain,and ultimately activates the transcriptional factors BZR1 and BES1 that are unknown to function in the BR signaling pathway to regulate the biological processes of tapetum formation.The main conclusions obtained are as follows:1.The intracellular domain of EMS1 and BRI1 can be replaced with each other.The transgenic complementation experiments showed that a chimeric receptor of BRI1-EMS1 using the extracellular domains and intracellular of BRI1 and EMS1,respectively,can replace the intracellular domain of BRI1 and completely maintain BRI1 biological functions.However,other receptor-like kinases could not be effectively exchanged,indicating that the functional replacement is specific.It is confirmed by genetic,biochemical,molecular and other experiments that the function of the chimeric receptor BRI1-EMS1 is completely dependent on the downstream signal transduction pathways of BRs.Conversely,Transgenic plants with the chimeric receptor EMS1-BRI1 has functioned as EMS1,suggesting that the downstream signaling pathways are similar for BRI1 of EMS1.2.The EMS1-TPD1 signal and the BRI1-BR signal can be partially replaced by each other.Ectopic co-expression experiments confirm that the EMS1-TPD1 signal can partially complement the BRI1-BR signal,which is independent of BR.Conversely,the BRI1-BR signal can also partially complement the EMS1-TPD1 signal.Biochemical experiments show that ectopic expression of EMS1&TPD1 can dephosphorylate and activate the transcription factor BES1.Taken together,EMS1-TPD1 signal can activate the transcription factor of the BR signal,and the BRI1-BR signal can determine the fate of the tapetum cells and produce a tapetum,implying their functional redundancy.3.BZR1 and BES1 function downstream of the signal of EMS1-TPD1.The mutants bzrl-1D and bes1-D,which are continuously activated with or without the BR signal,are able to restore the emsl,tpdl,and serk1serk2 mutants,and the EMS1 signal-associated mutants are regenerated into a tapetum to form a large number of fertile pollen and fruity siliques.The bzrl-1D and besl-D driven by the EMS1 promoter can also recover emsl,suggesting that the tapetum can be produced by activating the bzr1-1D and besl-D in the tapetum.Co-expression of EMS1&TPD1 did not restore the bin2-1D mutant,indicating that BIN2 is negative regulator located downstream of the EMS1-TPD1 signal.4.EMS1 and BRI1 control their biological processes through differential gene expressions.bril-116 emsl double mutant shows additive effects of the ems1 and bril-116 single mutant,indicating that the physiological processes of the two signal regulation were not overlapped.EMS1 driven by the BRI1 promoter could not restore emsl,and fluorescence study reveals that BRI1 is not significantly expressed in the tapetum,confirming that BRI1 and EMS1 control different biological phenotypes through differential expression patterns.Combining the above evidences of genetics and biochemistry,this study uncovers a cascade of EMS1 downstream signal transduction pathway for the first time.EMS1 determines the tapetum cell fate by activating the downstream transcription factors BZR1 and BES1 shared with BRI1.At the same time,it reveals the molecular mechanisms by which different signaling systems activate the same signaling pathway and regulate different biological processes.