Molecular Mechanism Research Of Rice Biological Clock Gene OsLUX Regulates Heading Date

Nearly 24 h of endogenous rhythm is generated to optimize internal processes that aim to cope with changes in the external environment and provide a fitness advantage for plants,especially,the heading date of rice,which is greatly affected by light and temperature factors,the rhythm oscillation of rice endogenous biological clock is critically important to ensure the growth and development and high and stable yield.In this study,we identified a circadian gene OsLUX by the Mutmap method,defect in OsLUX causes an extremely late heading phenotype under NLDs in Hangzhou and NSDs in Hainan.This paper focuses on identifying OsLUX,the expression patterns,the effects on other biological clock genes and flowering-related genes,interactive protein verification,the regulation of downstream target genes,and the construction of genetic materials.The research details are as follows:1.The phenotype identification and cloning of elh1We screened an extremely late heading mutant,elh1,from our rice mutant library generated by ethyl methanesulfonate-induced in Nipponbare.The mutant displayed an extremely late heading phenotype under NLDs in Hangzhou and NSDs in Hainan.OsLUX,an ortholog of Arabidopsis LUX,has a stop-gain mutation（CAG to TAG）at position 376 in elh1.To verify the possibility of whether OsLUX is the target gene,genetic complementation assay was carried out with a full-length genomic fragment from Nip to introduce into the elh1 mutant.The transgenic plants could fully rescue the extremely late heading phenotypes.When a p Actin1::OsLUX construct was transferred to the elh1 mutant,the transgenic plants showed a normal heading phenotype.The oslux mutants gained by CRISPR/Cas9 displayed a similar phenotype to the elh1 mutant,indicating that the mutation in OsLUX is responsible for the extremely late heading phenotype of elh1.In addition,we evaluated two other mutant alleles of OsLUX（elh2/oslux-2 and elh3/oslux-3）.The elh2 also causes a stop-gain mutation（TGG to TGA）at position 369,which had an exceptionally late heading phenotype similar to elh1.In contrast,the elh3 causes amino acid substitution at position469（CTC to TTC）,which resulted in Leu to Phe,and exhibited 34.4 days and 36.9 days later heading compared to Nip under NLDs in Hangzhou and NSDs in Hainan,respectively.2.The spatio-temporal expression pattern of OsLUXRT-q PCR and GUS activity analysis assays revealed that OsLUX was constitutively expressed among different tissues preferentially accumulated in leaf blades.Next,we examined the diurnal expression pattern.The results showed that OsLUX transcripts were increased during the light period and peaked in the evening（ZT=12）,subsequently reduced to the lowest level at dawn（ZT=0）under LDs and SDs.However,the rhythm and amplitude of OsLUX transcripts were slightly decreased when transferred to continuous light conditions.Still,they declined sharply when shifted to continuous dark conditions.Moreover,the expression waveform or amplitude of OsLUX was slightly higher in elh1 than Nip,probably due to the negative feedback regulation of OsLUX,as shown in Arabidopsis.We found the native OsLUX transcription levels were sharply reduced in35S::3×flag:OsLUX,which showed that OsLUX participates in a feedback loop.3.OsLUX participates in maintaining the circadian rhythm and regulating heading in riceTo explore whether OsLUX was involved in maintaining the circadian rhythm in rice,we examined the rhythm expression of the clock genes in elh1 and Nip,a higher amplitude of the rhythmical pattern of OsPRR37,OsPRR73,OsPRR59,OsPRR95,and OsPRR1 and low amplitude of the rhythmical pattern of OsCCA1 in elh1.Still,in continuous light conditions,the rhythm expression of these genes showed an arrhythmic oscillation.A Y1H assay showed that OsLUX facilitated the Lac Z expression of OsPRR1,OsPRR59,and OsPRR95,showing that OsLUX might directly or indirectly modulate the expression of these rhythm genes.The expression levels of flowering-time-related genes were also monitored in Nip and elh1 mutants via RT-q PCR under LDs and SDs,Ehd1 and Hd3a transcripts were almost invisible in elh1 mutants.However,the OsGI,Hd1 and Ghd7 transcripts significantly increased in elh1 mutants.4.OsLUX forms the OsEC complex through the recruitment of OsELF3-1 and OsELF4sTo investigate the molecular mechanism potential OsLUX regulates heading,we explored whether rice forms an EC complex similar to Arabidopsis.In rice,there are three orthologs of At ELF4:OsELF4-1,OsELF4-2,and OsELF4-3,and two orthologs of At ELF3:OsELF3-1 and OsELF3-2.Through yeast two-hybrid and luciferase complementation assays,we confirmed the physical interactions between OsLUX and OsELF4s with OsELF3-1 and OsELF3-2,respectively.OsELF3-1 is involved in circadian rhythm regulation and promotes heading,while OsELF3-2’s predominant role is immunity regulation.Consequently,we focused on whether OsELF3-1mediates flowering through the rice EC complex.Through Y2H assay,we found that the C-terminal（amino acids 503-760）and the middle region（amino acids 305-519）of OsELF3-1 could mediate a physical interaction with OsLUX and OsELF4s,respectively.But OsLUX and OsELF4s were unable to interact with each other.The interaction was also substantiated by pull-down,luciferase complementation assays and Bi FC.Investigating the triple complex’s subcellular localization found that OsLUX protein was located in the nucleus,cytosol,and membranes,and OsELF3-1 and OsELF4s were localized to the nucleus.Bi FC and co-localized assays showed that the interaction between OsLUX with OsELF3-1 and OsELF4s had accumulated in the nucleus.This observation indicates that OsELF3-1 alters the subcellular localization of OsLUX and promotes OsLUX translocation into the nucleus.The cell fractionation and immunoblotting assays confirmed that OsLUX was detected in the cytosol,membranes,and nucleus fraction.Coexpressed OsLUX and OsELF3-1 found both OsLUX had accumulated in the nucleus fraction.In Arabidopsis,ELF4promotes the nuclear localization of ELF3,to explore the effect of OsELF4s on the interaction between OsELF3-1 and OsLUX in rice.The luciferase complementation assay showed that the relative luciferase activity observed for the OsELF3-1–OsLUX interaction was significantly increased when OsELF4s were co-expressed with OsELF3-1 and OsLUX,suggesting that OsELF4s could enhance the interaction of OsELF3-1 and OsLUX.5.The genetic relationship of OsEC（OsELF4s–OsELF3-1–OsLUX）complexTo verify the genetic relationship between the OsELF4s–OsELF3-1–OsLUX trimer components,CRISPR/Cas9 was used to generate null mutants for each of the three genes.Under both NLDs in Hangzhou and NSDs in Hainan,the elf4-1 has no difference in heading.The elf4-2delay heading about 5.8 days under NLDs in Hangzhou,but the elf4-2 and elf4-3 mutants flowered slightly later than Nip under NSDs in Hainan.The mutation in OsELF3-1 resulted in 22.1-d and18.2-d later heading than Nip under NLDs and NSDs,respectively.We obtained the oslux-1oself3-1 double mutant through screening F₂ plants from the hybridized elh1 and oself3-1.The oslux-1 oself3-1 double mutant resembled the elh1 mutant,exhibiting an extremely late heading phenotype,and the late heading phenotype of oself3-1 oself4s plants resembles the oself3-1 single mutant.The genetic assay suggested that a compromised flowering phenotype results upon mutation of any component of the OsEC（OsELF4s–OsELF3-1–OsLUX）complex.6.OsEC complex inhibits the expression of Hd1 and Ghd7 through OsLUX.In Arabidopsis,LUX binds to the LBS motifs in the promoters of the output genes.We found the putative LBS motifs of LUX in the promoter of Hd1 and Ghd7.To investigate whether Hd1 and Ghd7 were the direct downstream target genes of OsLUX involved in regulating rice heading.The EMSA and Ch IP-Seq assays indicated that OsLUX could bind to the LBS site on the promoter of Hd1 and Ghd7 in vivo and in vitro.A relative transcriptional activity assay showed that OsLUX has a suppression effect on Hd1and Ghd7.To determine the effects of the OsEC complex on transcriptional repression of Hd1 and Ghd7,we performed a LUC transient transcriptional activity assay,the results indicated that the OsELF3-1-OsLUX dimer complex could enhance the suppressive activities of OsLUX to Hd1,and the OsELF4s–OsELF3-1–OsLUX complex was further strengthened the inhibition of Hd1.There is a parallel case that the OsEC complex has a similar suppression effect on Ghd7.Taken together,these results indicate that the OsEC complex through OsLUX directly binds to Hd1 and Ghd7 promoters and suppresses their expression.To explore the genetic relationship between OsLUX with Hd1 and Ghd7.We obtained the oslux-3 hd1double mutant through generated a null mutation of Hd1 by CRISPR/Cas9 against the background of elh3,and the oslux-1 ghd7 double mutants via screening F₂ plants from the F₁（elh1×ghd7）.Knock-out Hd1 and Ghd7 neutralized the repression of oslux on rice heading,which the oslux-3hd1 and oslux-1 ghd7 double mutants restored the extremely late heading phenotype of the elh3 and elh1,respectively.The genetic data suggest that OsLUX acts upstream of Hd1 and Ghd7 to regulate rice heading.7.OsLUX is located downstream of PHYBTo explore the genetic relationship between OsLUX and PHYB,we gained the oslux-1 phyb double mutants via screening F₂ plants from the F₁（elh1×phyb）.Under NLDs in Hangzhou,phyb mutants showed an early heading phenotype,while oslux-1 phyb showed a late heading phenotype compared with phyb mutants and an early heading phenotype compared with elh1 mutant.At the same time,RT-q PCR analysis showed no significant difference in the expression level of PHYB in elh1 mutant.But the OsLUX transcripts were increased in phyb mutant,indicating that OsLUX acts downstream of PHYB to regulate rice heading.Collectively,the OsEC（OsELF4s–OsELF3-1–OsLUX）target suppresses the expression of Hd1 and Ghd7 directly through OsLUX to modulate photoperiodic flowering in rice.The OsEC–Hd1/Ghd7 molecular module provides the genetic targets for crop genetic improvement.