Molecular Mechanism Of Arabidopsis NF-YA8 In Regulating Juvenile-to-adult Transition And Flowering Time

Higher plants undergo various phase transitions throughout their life cycle.Among them,the phase changes of vegetative(juvenile-to-adult)and flowering(vegetative-to-reproductive)are crucial for reproductive success and survival of higher plants.As a key component of the age pathway,microRNA156(miR156)and its target gene SQUAMOSA PROMOTER BINDING PROTEIN-LIKE(SPL)genes are master regulators that determine vegetative phase changes and flowering time regulation.The abundance of miR156 gradually decreases as plant age increasing,thus allowing accumulation of SPL mRNAs at adult stages.The accumulation of SPL promotes the juvenile-to-adult transition and flowering time.In addition,miR156 expression is also inhibited by sugar signaling,however,the specific regulatory mechanism is unclear.Nuclear factor Y(NF-Y)is an evolutionarily conserved trimeric transcription factor complex present in nearly all eukaryotes.The heterotrimeric NF-Y complex consists of three subunits,NF-YA,NF-YB,and NF-YC.Although Arabidopsis NF-YA,YB,YC play an important role in the regulation of flowering,it is still unclear whether they are involved in age-dependent flowering pathway.In this study,we investigated the phenotype of Arabidopsis NUCLEAR FACTOR Y A8(NF-YA8)transgenic plants and found that overexpression of this gene can inhibit the juvenile-to-adult transition and flowering time,which is similar to the phenotype of miR156 overexpressing plants.Based on the transcriptome data of NF-YA8-3MYC transgenic plants,we found that NF-YA8 can regulate the expression of flower-related genes in many age pathways and integration pathways,indicating that NF-YA8 may regulate plant growth and development through two pathways.By using several strategies of molecular biology,biochemistry and genetic manners,we analyzed the molecular mechanism of NF-YA8 regulating Arabidopsis juvenile-to-adult transition and flowering time.The main findings are as follows:(1)We first generated the transgenic NF-YA8-3MYC overexpression lines.Compared with WT No-0 control plants,NF-YA8-3MYC transgenic lines(A8-O1,A8-O7 and A8-O8)dramatically delayed flowering under both LD and SD growth conditions.In addition,we found that NF-YA8-3MYC transgenic plants displayed later marginal serration and abaxial trichomes compared to WT plants under LD growth conditions,indicating that Arabidopsis NF-YA8 inhibit the juvenile-to-adult transition and flowering time.(2)We used transcriptome and RT-qPCR experiments to explore the downstream genes regulated by NF-YA8.The results showed that NF-YA8 could regulate the transcription of many age pathway-related genes(SPL3,SPL4,SPL5,SPL5,and SPL15)and integration pathway flowering-related genes(FT,SOC1,FLC,and AG).These results suggested that NF-YA8 negatively regulates flowering time by affecting the expression of flowering integrators and age-dependent flowering pathways.(3)Promoter sequence analyses revealed that multiple NF-Y transcription factor binding cis-elements(CCAAT)were identified within 2 kb upstream of the transcription start site(TSS)for MIR156 s and SPLs.Next,we performed transient expression assays using dual-luciferase(LUC/REN)reporters in Arabidopsis protoplasts.The results showed that NF-YA8 could activate the transcription of MIR156 A,C,D,and E.However,when the CCAAT-box was mutated,this activation obviously lost.These findings suggest that NF-YA8 directly binds to MIR156 promoters and activates their transcription.(4)Genetic results showed that A8-O1 plants presented a delayed juvenile-to-adult transition and late flowering phenotype.MIM156(miR156 target mimics,which reduced miR156 activity)and A8-O1 MIM156 plants exhibited an earlier juvenile-to-adult transition and early flowering phenotype,indicating that miR156 may genetically locate the downstream of NF-YA8 in the regulation of juvenile-to-adult transition and flowering time.(5)To investigate the spatial and temporal regulatory mechanisms of the NF-YA8—MIR156s transcriptional cascade,RT-qPCR and GUS activity analyses were used.We found that the NF-YA8 transcript levels dramatically decreased with increasing leaf age.To further verify whether NF-YA8 protein largely increased in juvenile stage leaves,we generated NF-YA8p::NF-YA8-LUC transgenic plants,and found that accumulation of the NF-YA8-LUC fusion protein was significantly increased in juvenile-stage leaves,but was drastically reduced with increasing development age.Previous studies demonstrated that the expression of MIR156 A and MIR156 C were also regulated by sugar signals.We performed sugar treatment experiments and confirmed that the transcript and protein levels of NF-YA8 were strongly repressed by sugar.These results indicate that NF-YA8 may serve as a signal hub of sugar signal and age signal to regulate MIR156 transcription.(6)In addition to the age pathway,our transcriptome data showed that genes associated with the integration pathway were significantly changed in NF-YA8-3MYC plants compared to WT plants.Our RT-qPCR analysis further confirmed that NF-YA8 could inhibit FT expression.Further ChIP-qPCR and transcriptional activation experiments confirmed that NF-YA8 directly bound to FT promoter and suppressed its transcription.(7)Yeast two hybrid assays showed that NF-YA8 interacted with ELF4.BiFC,and Co-IP assays further confirmed the interaction occurring in vivo.(8)Genetic results showed that elf4 mutant had early flowering phenotype,while the NF-YA8-3MYC overexpressing transgenic plant had a clear late flowering phenotype.The A8-O1 elf4 plant showed a clear late flowering phenotype similar to A8-O1 overexpressing plants,which indicates that NF-YA8 may genetically locate the downstream of ELF4 in regulation of flowering time.(9)We performed transient expression assays using dual-luciferase(LUC/REN)reporters in Arabidopsis protoplasts,and found that NF-YA8 or ELF4 alone significantly repressed the expression of LUC reporters.However,when NF-YA8 and ELF4 were co-expressed,the reporter activity was dramatically reduced.The transient expression assays in N.benthamiana leaves also confirmed that ELF4 and NF-YA8 may coordinately inhibit the transcription of FT,thus repressed flowering time in plants.In summary,we investigated the molecular function of Arabidopsis NF-YA8 in vegetative phase changes and flowering time.NF-YA8 integrates age and sugar,directly triggering miR156-SPL-regulated processes and repressing the juvenile-to-adult transition.In addition,ELF4 and NF-YA8 can coordinately inhibit the transcription of FT and inhibit flowering time to ensure plant flowering at the right development phase and season.