| Flowering time is one of the most fundamental factors determining the distribution and final yield of rice.For Ehd1,a type B response regulator that plays a key role in rice photoperiod regulation,its expression has been reported to be influenced by multiple flowering promoters or repressors upstream,but its potential regulators remain to be identified.Previous studies showed that the basic leucine zipper transcription factor bZIP71 is a suppressor of rice flowering,and the genetic analysis showed that bZIP71 function is independent of Heading date2(Hd2),Hd4,and Hd5.Instead,by interacting with two members of the polycomb inhibitory complex 2(PRC2),SDG711(SET domain group protein 711)and FIE2(Fertilization independent endosperm 2),regulating the H3K27me3 levels of Ehd1.The present study identified bZIP65 is the closest homologs to bZIP71,and it was speculated that bZIP65 may also be involved in heading date regulation in rice.To dissect the biological functions of bZIP65,we generated the bZIP65 OE and bzip65 single mutants in SJ2 background.Further through the study of the expression characteristics,we found that bZIP65 present opposite optoperiodic rhythmicity with key factor Ehd1,suggesting that its function may depend on Ehd1.Further analysis of its molecular mechanism by using yeast two-hybrid,yeast single hybrid,gel shift assay,protoplast transient transformation system and ChIP-qPCR and other biochemical manners.The main study results are presented as follows:1.bZIP65 is an ortholog of bZIP71 in the bZIP family evolutionary relationship,and overexpression of bZIP65 in the SJ2 background delayed rice flowering,while bzip65 mutant have similar flowering time to SJ2 in both long day(LD)and short day(SD)conditions.2.Studies on the expression characteristics of bZIP65 found that it had transcriptional activation activity and showed tissue-specific expression,with the highest expression in rice roots.Photoperiodic rhythmic analysis showed that bZIP65 and Ehd1 showed the opposite expression pattern in both LD and SD conditions.3.The yeast two-hybrid results showed there was no direct interaction between bZIP65 and Ehd1.4.We further confirmed that bZIP65 was able to bind to the promoter regions of Ehd1 and transcriptionally inhibited Ehd1 expression by biochemical manners including yeast single hybrid,EMSA and transient expression assay in rice protoplasts.5.ChIP-qPCR using H3K27me3 antibody showed that bZIP65 overexpression enhanced the modification level of H3K27me3 of Ehd1.In conclusion,we found a new gene bZIP65 that regulates heading date in rice.Further analysis of its molecular mechanism showed that bZIP65 inhibited Ehd1 expression by increasing H3K27me3 level of Ehd1,and thus inhibited rice flowering. |
