Identification And Functional Analysis Of Natural Variation Of Flowering Regulator SSF In Arabidopsis Thaliana

Flowering is an important transition from vegetative to reproductive stage in the lifecycle of plants.Flowering time is a complex trait and many factors have been reported to be involved in flowering time control.However,to date,many regulators remain undiscovered,and their biological functions have not been characterized.In this study,we screened the natural variation of flowering time in Arabidopsis thaliana populations using bioinformatics,genetics,molecular biology and biochemistry approaches,and identified a functional natural variation in SSF(Sister of FCA)gene.Then we investigated the molecular mechanism of this natural variation in flowering time regulation.The major results are listed as below:1.We determined FLC expression before and after vernalization treatment in 102Arabidopsis ecotypes and performed GWAS analysis on this data.Then we crossed two accessions Col-0 and Lov-1,which have significant differences in flowering time,and obtained an F₂ population containing 284 individual plants for QTL mapping.Three QTLs were identified,and one of them co-localizes with a SNP signal at the end of chromosome2 in our GWAS analysis using natural Arabidopsis thaliana accessions.2.Bioinformatics analysis suggested that SSF was a candidate gene in the Chr-2 QTL interval.The ssf mutants were early flowering and has lower FLC expression compared with wild type control,suggesting that SSF is a positive regulator for FLC regulation.Tissue expression patterns and subcellular localization demonstrated that SSF was localized in the nucleus,and highly expressed in floral organs.Genetic pathway analysis suggested SSF as a novel factor involved in the autonomous pathway regulating flowering time.Furthermore,transcriptional activity and chromatin immunication precipitation assays provided direct evidence that SSF regulated FLC at the transcriptional level.The transgenic plants containing allelic variations of SSF(SSF414N/ssf-2 and SSF414D/ssf-2)showed significant FLC expression and flowering time differences.3.Using protein IP-MS method,we screened an SSF interacting protein,a ubiquitin E3 ligase-CUL1.This interaction was validated by Y2H,BIFC,in vitro Pull-down and in vivo Co-IP approaches,and further analysis showed that SSF allelic variations affected the strength of its interaction with CUL1.4.FLC expression was down-regulated and FT was upregulated in cul1-1 mutants,which led to an early flowering phenotype.CUL1 was proved to promote SSF protein degradation by qualitative and quantitative protein ubiquitination assays.And our results showed that CUL1 could also interact with SSF homolog FCA and inhibit it protein accumulation.In addition,the non-synonymous mutation N414D affected the degree of SSF protein ubiquitination,thereby regulating SSF protein stability and affecting flowering time.5.There is significant climate difference in the north and south of Sweden.We found that the SSF414D majorly exists in N.Sweden and SSF414N in S.Sweden,which imply that the SSFN414D variation may have played a role in local adaptation of Arabidopsis plants.In addition,evolutionary analysis of SSF in angiosperm showed that SSF only exist in monocot palnts and its homolog FCA exists in both monocot and dicot plants,the SSFN414D emerged after the divergence of monocot and dicot plant,the SSF414N only exits in some Arabidopsis accessions.Overal,we show a novel flowering time regulator SSF and reveal its role in flowering time regulation.SSF interacts with protein ubiquitination pathway,and the SSFN414D protein can be degradaded differentially in ubiquitination depedent manner.We finally explored the possible role of the natural variation of SSF in plant local adaptation.