Study On Molecular Mechanism Of Heading Date Under Long-Day Conditions Mediated By HAF1 In Rice

Rice is an important food crop and model plant for genomic research among cereal crops.Heading date is one of the most important factors associated with grain yield,determining the seasonal and regional adaptability of varieties in rice.Although rice is a short-day plant,artificial breeding enables accessions to flower and harvest under long-day conditions,which expands the regional distribution of rice accessions.Rice planting region spreads widely in China,covering form Hainan to Heilongjiang provice.During the last decades,the molecular network of photoperiodic flowering pathway in rice has been preliminarily established.A large number of transcription factors regulating the heading date were identifed and cloned in rice.We previously demonstrated that HAF1,a C3HC4 RING domain-containing E3 ubiquitin ligase,is essential to precisely modulate the timing of Hd1 accumulation and to ensure appropriate photoperiodic responses under short-day conditions.However,how HAF1 mediates flowering under long-day conditions(LDs)remains unknown.In this study,we showed that OsELF3(EARLY FLOWERING3)and Ghd7(Grain number,plant hight and heading date 7)are the direct substrates of HAF1 for ubiquitination in vitro and in vivo.Genetic analysis revealed the molecular mechanism between HAF1 and its substrates OsELF3 and Ghd7 in regulating flowering under LDs.The main results were described as follows:1.HAF1 maintains the phase of OsELF3 accumulation by mediating the ubiqutination and degradation of OsELF3Yeast two-hybrid assay demonstrated that HAF1 interacts with OsELF3 in yeast cell and the C terminal region of OsELF3 is required for the interaction with HAF1 and formation a homodimer by itself.Bimolecular fluorescence complementation(Bi FC)and pull-down assays further verified the interaction relationship between HAF1 and OsELF3 and the homodimer formation of OsELF3;In vitro ubiquitination and cell-free degradation assays showed that HAF1 ubiquitinates OsELF3 in vitro and mediates OsELF3 degradation through 26 S proteasome.In vivo degradation assay verified that HAF1 degrades OsELF3 in planta by 26 S proteasome-dependent pathway;Immunoblot analysis using anti-OsELF3 antibody showed that OsELF3 accumulates in a rhythmic pattern.In haf1,the phase of OsELF3 rhythmic accumulation patterns was disturbed.These results indicate that HAF1 is indispensible for maintaining the circadian rhythm of OsELF3 accumulation in rice flowering pathway.2.HAF1 affects the molecular network of heading date associated with OsELF3We analysised expression pattern of heading date associated genes in haf1,oself3,haf1 oself3 and WT plant under LD conditions.The transcript levels and diurnal expression phases of OsGI,Hd1,and Ghd7 did not obviously differ between haf1 and WT plants.Also,HAF1 had no effect on the expression pattern of OsLHY,OsPRR1,OsPRR37,or OsPRR59.Thus,we propose that HAF1 affects the function of OsELF3 mainly on the protein level but does not affect its diurnal transcriptional pattern.3.Interacting relationship between HAF1 and OsELF3 encoding by OsELF3 alleles affacts the contribution of OsELF3 alleles to geographical distribution of rice accessions There are abundant natural variations within OsELF3 locus in rice germplasm resources.Among these variation sites,the second SNP(A/G)introduces the amino acid variation from leucine to serine at residue 558.GWAS of heading date showed that the second SNP(A/G)at position 5191 significantly associates with the heading date,OsELF3(L)type accessions flower oberviously earlier than OsELF3(S)type,and OsELF3(L)type accessions distribute in higer latitude regions and OsELF3(S)in lower latitude regions.Protein interaction and cell-free degradation assays showed that HAF1 interacts with OsELF3(L)and mediates the degradation of OsELF3(L),but HAF1 does not interact with OsELF3(S)or mediate its degradation,indicating that the mechanism of OsELF3 protein degradation is different among accessions.4.The genetic relationship between HAF1 and OsELF3Because haf1 oself3 double mutant flowered as late as oself3,obviously later than haf1 mutants and WT plants,indicating that OsELF3 is epistatic to HAF1 in rice flowering pathway under LDs;when OsELF3 was overexpressed in haf1 background,the earlier heading date of OsELF3-OX/haf1 double mutant as well as OsELF3-OX plants compared to haf1 plants further indicated that OsELF3 is epistatic to HAF1.5.HAF1 interacts with Ghd7 and mediates its ubiquitination and degradationProtein interaction assays showed that HAF1 interacts with Ghd7,another factor associated with heading date under LDs.Two regions of Ghd7: aa(101-150)and aa(190-223)is indispensible for its interaction with HAF1.In vitro ubiquitination and cell-free degradation assays showed that HAF1 ubiquitinates Ghd7 and mediates degradation of Ghd7 by 26 S proteasome.6.The genetic relationship between HAF1 and Ghd7,OsELF3 and Ghd7We investigated the heading date of haf1,oself3,ghd7,haf1 ghd7 and oself3 ghd7 plants under LD conditions,showing that Ghd7 is epistatic to HAF1 and OsELF3 in rice flowering pathway under LDs.