Functional Analysis Of DHD4 And EH7 In Controlling Heading Date In Rice

The heading date is one of the important agronomic traits in rice,which determines the adaptability of rice to regions and rice yield.Identification of genes on heading date and establishment of regulatory network can provide gene reserve and theoretical support for rice breeding.In this study,we identified two key genes DHD4 and EH7 in regulating heading date and analyzed their functions.1.Functional analysis of DHD4 in regulation of heading date in riceIn previous studies,mutants and near-isogenic lines were used to identify important genes on heading date by map-based cloning.Via this method,a number of major-effect genes on heading date were identified(such as Ghd7,DTH8 and Hdl).However,the minoreffect genes with low effect on heading date are difficult to identify and clone.In modern breeding,with the progress and development of rice breeding improvement,the minor-effect genes are of great significance for the fine-tuning of rice agronomic traits.In this study,we identified a minor-effect factor DHD4 in regulation of rice flowering by reverse genetics and studied its molecular mechanism.The main results are as follows:(1)Overexpression of DHD4 in Kitaake or Nipponbare both showed significantly delayed flowering.The DHD4 knockout lines or RNA interference lines in the background of Nipponbare showed early flowering about 3-4 days without a notable yield penalty compared with WT under natural long day(NLD)conditions.Sequence analysis revealed that DHD4 is a COSTANS-like(COL)protein containing a conserved CCT domain.Tissue expression analysis showed that DHD4 is mainly expressed in shoot apex,buds and panicles,but is lower in leaves,roots,and sheaths.Further biochemical experiments showed that DHD4 localizes to the nucleus and can form homologous complex in yeast cells.(2)Via yeast two-hybrid screen library,OsFD1 was identified to interact with DHD4.Further,the interaction between DHD4 and OsFD1 was confirmed by pull-down,Co-IP and FLCI assays.To verify the function of OsFDl in rice flowering,the CRISPR/Cas9 gene editing system was performed to produce Osfdl and dhd4 Osfd1 in Nipponbare.The Osfdl mutant showed significantly delayed flowering under NLD and NSD conditions.This indicates that OsFD1 plays a key role in regulation of flowering pathways in rice.The heading date of the dhd4 Osfd1 were significantly later than wild type and dhd4,while consistent with the phenotype of Osfd1,indicating that DHD4 and OsFD1 function in the same pathway in regulation of rice heading date.(3)The pull-down of competitive binding assay was performed to analysis the relationship among DHD4,GF14c and OsFD1.The results showed that with the increase of DHD4 protein,the interaction of OsFD1-GF14c gradually decreased.Similarly,as the GF14c protein increases,the interaction of OsFD1-DHD4 is also gradually affected.These data indicated that DHD4 competes with GF14c to bind OsFD1.The OsFD1-GF14c interaction is the key to form FAC complex.Therefore,whether DHD4 will ultimately affect the formation of FAC.Similarly,the pull-down of competitive binding assay showed that with the increase of DHD4,GF14c and Hd3a,which were bound by OsFD1,gradually decreased,indicating that the OsFD1-GF14c-Hd3a interaction gradually weakened,further confirming that DHD4 affects the formation of FAC complex.Transient transformation experiments in tobacco and rice protoplasts confirmed that the addition of DHD4 reduced the transcriptional activation activity of FAC complex on OsMADS15 promoter.Further,RT-qPCR analysis showed that the expression of OsMADS14,OsMADS15 were markedly decreased in the DHD4 overexpression lines,but increased in the dhd4 mutant.Overall,DHD4 inhibits the expression of OsMADS14,OsMADS15 by affecting the formation of FAC complex.(4)To verify the genetic relationship between DHD4,OsMADS14 and OsMADS15,we overexpressed OsMADS14 and OsMADS15 in the background of DHD4-OE,respectively.Phenotypic identification showed that the heading date of both double overexpressed lines were earlier than that of DHD4-OE,indicating that overexpression of OsMADS14 and OsMADS15 can make DHD4-OE flowered earlier.These results confirmed that DHD4 functions upstream of OsMADS14 and OsMADS15 to regulate rice heading date.2.Functional analysis of EH7 in regulation of heading date in riceHd1was reported to have dual regulation in controlling rice heading date which inhibits flowering under long-day conditions,while promotes flowering under short-day conditions.Two molecular mechanisms were reported for the flowering inhibition of Hd1 under longday conditions.On the one hand,Hdl interacts with Ghd7 to inhibit the expression of Ehdl and then delays flowering.On the other hand,Hdl also forms complex with DTH8 to reduce the expression of Hd3a and delays flowering.Ghd7 and DTH8 are two key proteins in regulating flowering,and simultaneously affect plant height and crop yield.However,the relationship between Ghd7 and DTH8 protein were not clear.In this study,an early flower mutant eh7 was identified by forward genetics,which showed early flowering under longday conditions.By phenotypic identification,primary mapping and complementation experiments,Ghd7 was confirmed as the candidate gene of EH7.Further,the relationship between Ghd7 and DTH8 were analyzed.The main results are as follows:(1)Under LD conditions,eh7 flowered earlier than wild type,about 25 days.While,under SD,there were no differences between the mutant and wild type.Primary mapping showed that EH7 localizes to the centromere region of chromosome 7.Further study confirmed that the transposon insertion in the 5'UTR region of Ghd7 affected the transcriptional expression of Ghd7 and led to the early flowering phenotype of eh7 mutation.(2)Rhythm expression analysis of genes in controlling heading date showed that the expression of Ehdl and its downstream florigen genes Hd3a/RFT1 were significantly increased in eh7,indicating that Ghd7 delays flowering by inhibiting the expression of Ehd1.Further,molecular experiments showed that Ghd7 can interact with DTH8 to form heterologous complex.Based on the interaction between Hdl with Ghd7 and DTH8,this study further confirmed that Ghd7 can form poly complex with Hdl and DTH8,which provides new clues for further explaining the dual functionality of Hd1.