Molecular Mechanism Of The E1-GmSPL3b Module In Soybean Photoperiod Response

The photoperiod sensitivity of soybean restricts the planting range of soybean varieties and restricts the popularization of superior soybean varieties.E1 gene is the core of regulating soybean photoperiod response and is a unique transcription factor of leguminous crops,but the downstream pathway of its regulation is still unclear.In the previous study,our laboratory discovered a gene GmSPL3b regulated by E1.The expression of GmSPL3b is up-regulated in E1 overexpressing plants.The results in Arabidopsis and rice show that the SPLs gene family is regulated by mi R156 and participates in the regulation of flowering through the age pathway,while the primary transcript of GmSPL3b does not contain the Gmmi R156 recognition site.Therefore,GmSPL3b is not the target gene of Gmmi R156 and is not regulated by it,indicating that GmSPL3b may regulate the growth period of soybean by participating in a new pathway.In addition,E1 is a unique transcription factor of leguminous plants,and"E1-GmSPL3b"is a unique photoperiod unit in soybean.The research on the regulation module,the fine regulation mechanism of the E1-GmSPL3b module is of great scientific significance for clarifying the molecular regulation mechanism of the photoperiod unique to soybean,and a breakthrough will be made in the regulation mechanism of soybean flowering.In this study,we explained the GmSPL3b was depressed by E1,and discovered two genes（GmMDE06 and GmMDE13）were regulated by GmSPL3b directly in vivo.The main findings are as follows:1.The up-regulated expression of GmSPL3b was detected in e1^CR,a loss of function plant,and we further determined that GmSPL3b expression could be inhibited by E1.Using a tobacco leaf transient expression system,E1 was found to repress the promoter activity of GmSPL3b.2.Analysis of the subcellular localization and transcriptional activation of GmSPL3b,a transcription factor encoding an SBP domain,revealed that it is ubiquitously expressed in cells and can promote the activation of other transcription factors and is a transcriptional regulator.3.Overexpressing GmSPL3b soybeans presented an early flowering phenotype,and GmFT2a/5a and GmMDE06 up-regulated expressions were detected in their leaves,whereas GmSPL3b did not present up-regulated expressions in plants overexpressing GmFT2a/5a and GmMDE06,which illustrated that GmSPL3b was located upstream of GmFT2a/5a and GmMDE06 to regulate their expressions.GmSPL3b,but not GmFT2a/5a,was shown to bind directly to the promoter of GmMDE06 by chip q PCR.4.Plants sense the light signal through the leaf,and flowering hormones transmit the light signal to the shoot apical meristem to induce flowering.The molecular mechanisms responding to the photoperiod are different in leaves and shoot apices,therefore,we further analyzed the transcriptome differences in the SAM of GmSPL3b overexpressing soybean,screened the genes regulated by GmSPL3b,and performed q PCR for verification,and found that gmspl3b promoted the expression of a large number of MADS-box class genes and flowering related genes such as LFY in the SAM.5.Through BiFC,LCI with CO-IP confirmed that the GmSPL3b would interact with GmFT2a/5a and GmFDL19.In soybean SAM,GmMDE06 and GmMDE13directly regulated by GmSPL3b were identified by Chip-q PCR.The direct binding site of GmSPL3b also contains cis-elements for SPL binding to FD transcription factors,further indicating that GmSPL3b may participate in GmFT2a/GmFT5a/GmFDL19complex formation and co-regulate downstream gene expression.In summary,this study further establishes that GmSPL3b is located downstream of E1,a photoperiod core gene,and refines the molecular pathway downstream of E1through the study of the fine molecular mechanism by which flowering is regulated by GmSPL3b.This innovative finding that GmSPL3b is a gene that can function simultaneously in leaves and shoot apices adds to the understanding of the photoperiod reaction mechanism in soybean and provides new ideas for molecular design breeding.