The Function Of Nuclear Localization Signal Of SPL9 Transcription Factor In Arabidopsis

The juvenile-to-adult phase transition is a critical developmental process in plants,it is regulated by the conserved microRNA-miR156 and its target genes SPLs?SQUAMOSA PROMOTER BINDING PROTEIN-LIKE?.SPLs are plant-specific transcription factors,which play important regulatory roles in plant growth and development,secondary metabolite biosynthesis,and response to stresses.In order to identify more regulatory factors involved in the miR156-SPL pathway,we screened an EMS?ethyl methylsulfonate?-mutagenized population,and we identified a mutant exhibiting a delayed vegetative phase change phenotype?delayed vegetative phase transition mutant 6,del6?.The mutation was cloned by map-based cloning and found to encode the SPL9 protein.Whole genome re-sequencing indicated that it is a G-to-A substitution at the 434bp from the start codon in the SPL9sequence,this causes a replacement of 145-arginine?Arg,R?by a glutamine?Gln,Q?.Sequence analysis revealed that the mutation was located in the conserved nuclear localization signal region?NLS?of the conserved SBP region.We conducted a series of studies using this mutant.The main results are as following:?1?Phenotype analysis shown del6 acts as delayed vegetative phase change mutant,including delayed abaxial trichomes formation,fewer leaf serrations,rounder leaf and quick leaf initiation.?2?Map-based cloning narrowed the candidate region between position 16291Kb and 19542Kb.Whole genome re-sequencing showed six different mutations within this candidate region,and one of them is located within the SPL9 coding region.There is a G-to-A substitution at the 434bp from the start codon in the SPL9 sequence,this causes a replacement of 145-arginine?Arg,R?by a glutamine?Gln,Q?within the SBP conserved domain.Therefore,the SPL9 gene is used as a mutation candidate gene.?3?In order to conform del6 acts as allele mutant of SPL9 gene,genetic complementation test was taken by crossing del6 to spl9-4.The vegetative phase change phenotype of del6 x spl9-4 F₁ plants were similar as del6 and spl9-4,which indicated del6 is a new functional deletion allelic mutation of the SPL9 gene.Future qRT-PCR detection result conform expression of pri-MIR172B,a direct target of SPL9,was down-regulated in del6.?4?Mutation of del6 changed the conserved 72th amino acid within the SBP domain.POSRTII analyzing indicated the basic amino acid-rich motif within the C-terminal of SBP domain function as NLS.They are three different NLS,a monopartite NLS 1,a monopartite NLS 2 and a bipartite NLS 1,and the 72th amino acid is necessary for all above NLS.?5?In this study,subcellular localization analyzing was taken to conform the effect of R-72-Q mutation in SPL9 nuclear import.35S::GFP?control plasmid?,35S::GFP-SPL9^WT?SPL9 CDS from WT?and 35S::GFP-SPL9^del6?SPL9 CDS from del6?plasmid were constructed,and transformed to Arabidopsis mesophyll protoplast,respectively.The detection results shown the control GFP distributes in nucleus and cytoplasm together,the GFP-SPL9^WTT fusion protein was imported into the nucleus,however,the GFP-SPL9^del6el6 fusion protein was distributed in the nucleoplasm and the cytoplasm,and the GFP signal in the cytoplasm is stronger than GFP.Above results indicated that the SPL9 transcription factor is a nuclear localization protein,and the SBP R-72-Q mutation in the del6 mutant lead nucleus import defect.?6?The SPL9 fragment of the WT and del6 mutants were cloned and transformed into WT plants,and made the gSPL9^WTT and gSPL9^del6el6 transgenic plants,respectively.gSPL9^WTT transgenic plants produced a precocious vegetative phase change phenotype,including precocious abaxial trichomes formation,more leaf serration,greener leaf color,however,the phenotype of gSPL9^del6el6 transgenic plants is similar to WT.This result conforms the defect of SPL9^del6el6 in del6 mutant.In summary,a new loss-of-function allelic mutant of SPL9,named as del6,was obtain in Arabidopsis thaliana,and this study uncovered the conserved C-terminal basic amino acid motif within the SBP functions as the NLS for SPLs.Mutation within the NLS disrupts the nucleus import and function of SPLs transcription factor.Our results will benefit for designing of gene edit site,and regulation of SPL9 and its orthologous genes?such as tomato SlySPL15,grape VvSPL9 and rice OsSPL14?.