The Epigenetic Regulator ULTRAPETALA 1 Suppresses De Novo Root Regeneration And Accelerates Flowering In Arabidopsis

The spatiotemporal patterns of gene expression play critical roles in plant development.In the developmental gene regulatory networks(GRNs),epigenetic regulation is an important mechanism to stabilize the gene expression patterns,which includes two main epigenetic mechanisms,DNA methylation and histone modifications.DNA modification usually leads to permanent gene silencing.In contrast,histone modifications are expression switches that regulate gene expression in different tissue and at different times,but the histone modification pattern can be reset.Among histone modification-based gene regulation,two groups of protein complexes catalyze antagonistic histone marks.One is called Polycomb group(Pc G),catalyzing the repressive marks H3K27me3 and H2Aubi;other complexes belonging to the Trithorax group(Trx G)catalyze activation-related marks such as H3K4me3 and H3K36me3.They were first discovered in Drosophila and later found widely spread among eukaryotic organisms including plants.ULTRAPETALA 1(ULT1)encodes a DNA-binding SAND box protein with functions in regulating meristem size in inflorescences and maintaining stem cell niche in roots in Arabidopsis thaliana.ULT1 interacts with the Trx G protein ATX1,which regulates the flower development gene AGAMOUS(AG).However,ULT1 interacts with the Pc G protein EMF1 regulating seed maturation via directly controlling ABI3 expression.ABI3 is involved in de novo root regeneration(DNRR).From these previous findings raises the question whether ULT1 is also involved in root regeneration.Furthermore,loss of ULT1 results in delayed flowering time,but the exact mechanism was still unclear.Therefore,this study focused on the control of DNRR and flowering time by ULT1 and tried to discover the epigenetic mechanisms of ULT1 during development.This study discovered several new findings:1.ULT1 inhibits DNRR by repressing auxin biosynthesis.Loss of ULT1 results in more adventitious roots from leaf explant in both aspects,rooting rate and rooting capacity.30 min after detached,ABI3 expression was not significantly changed between wildtype and ult1-3 mutant leaf explants.However,ERF109,encoding an activator of the auxin biosynthesis gene ASA1,and ASA1 itself were increased dramatically in ult1-3 mutant explants.At the same time,repressors of ERF109,which are SPL2,10,and 11,were not significantly changed.The rooting rate and capacity differences were seized when exogenous auxin was applied.The expression of other main auxin biosynthesis genes such as YUCCA 2(YUC2),YUCCA 4(YUC4)and YUCCA 6(YUC6)were not changed.These results suggest that ULT1 is a repressor of adventitious root formation by repressing ERF109 independently of SPL2,10 and 11.Hence,ULT1 could be part of a novel mechanism preventing in parallel to the SPL module over-proliferation of adventitious roots in leaf explants by inhibiting auxin bursts in response to wounding.Since ULT1 interacts with the Polycomb protein EMF1,the repression of ERF109 could be direct.Another possibility is that ULT1 as Trx G factor activates a transcriptional repressor of ERF109.2.ULT1 promotes flowering by the autonomous flowering pathway.Loss of ULT1 results in a delayed flowering time phenotype under both LD and SD.Compared with wild-type,the ratio of rosette leaf number under LD to SD indicates that ult1 mutants are not affected in the day-length pathway.After GA treatment,flowering time of ul1-3 mutants was accelerated,while vernalization experiments showed that after 3-6 weeks cold treatment,ult1 mutants flowered earlier than ult1 without cold treatment.These findings indicate that ult1 mutants are not disturbed in the day-length,GA and vernalization pathway,which,in turn,make ULT1 to a putative member of the autonomous pathway that represses FLC.In line with this,ult1-3 flc-5 double mutant showed a flowering time similar to wild-type and,therefore,the complete rescue of the late flowering phenotype in ult1-3 mutants.3.ULT1 represses the expression of FLC.RNA-seq data and RT-q PCR results showed that FLC was strongly elevated in ult1 mutants(14 and 21 DAG).RNA-seq also showed that FLC is the most misregulated gene in ult1-3.In contrast,other read-out gene of other flowering time pathways such as SHORT VEGETATIVE PHSAE(SVP),FLOWERING LOCUS M(FLM)or CONSTANS(CO)was not significantly changed.High FLC expression was confirmed by FLC::GUS staining in wild-type and ult1-3 mutants showing that FLC was also in earlier stages(4 and 10 DAG)higher and prolonged expressed in ult1-3,while in wild-type,FLC was broadly downregulated 10 DAG.Although total COOLAIR expression of 14 DAG seedlings was not different in wild-type and ult1-3,the proximal/distal COOLAIR ratio decreased.This decrease could also be found in one-day-old seedlings,whereas at 21 DAG,this difference was reversed.This indicates that ULT1 could be involved in activating and processing COOLAIR,which,in turn,results in silencing of FLC and COOLAIR.4.Loss of ULT1 resulted in increase of H3K4me3 but decreased of H3K27me3 at the FLC locus.In ult1 mutants,FLC gains H3K4me3 that is an activation-related mark around the transcription start site.The H3K27me3 levels,in both ult1 mutants,were reduced thought out the FLC gene body.In Y-2-H assays,the ULT1 bait interacted with CURLY LEAF(CLF),SWINGER(SWN)and MULTI-COPY SUPPRESSOR OF IRA4(MSI4,also named FVE),which are all PRC2 components,while FVE is also a classical member of the autonomous pathway.The interaction ULT1 with CLF and FVE was confirmed in luciferase complementary image assays.ULT1 also could bind to itself.Bioinformatics analysis regarding the hinge region between SAND and B-box like domain in ULT1 revealed a structural homology with 58.9% confidence between 42 residues of the uncharacterized region in ULT1 and the CW-type zinc finger domain of human MORC3.Notably,ult1-1 carries a single point mutation disrupting one of the CW-type zinc finger.These findings indicate that ULT1 could be a recruiter of PRC2,which in turn leads to H3K27me3-mediated gene silencing of FLC and COOLAIR,while the CW domain might be needed for the interaction between ULT1 and PRC2 and/or the FLC/COOLAIR gene locus.Taken together,this study shows that ULT1 inhibits DNRR by repressing auxin biosynthesis and promotes flowering by repressing FLC.ULT1 belongs to the autonomous flowering pathway.ULT1 can interact with the PRC2 components CLF,SWN and FVE that is an autonomous pathway member.Since loss of ULT1 leads to decreasing of H3K27me3 levels at the FLC locus,ULT1 likely recruits PRC2 to silence FLC by H3K27me3.However,changes in COOLAIR expression and ratio of splicing variants in ult1 mutants suggest that ULT1 might repress FLC expression additionally via activation and processing of COOLAIR.It will be helpful to elucidate the mechanisms of UTL1 involved in plant developments in the future.