Mechanism Of ZmCCT Regulating Photoperiod Response And Gibberella Stalk Rot Resistance In Maize

Maize is an important food crop,and plays a vital role in ensuring food security.The ability of responding to photoperiod is one of the key factors affecting growth period of maize,which limits the adaption zones and yield of different maize varieties.Gibberella stalk rot in maize is also a widespread soil-born diseases affecting the production.Previous studies have showed that ZmCCT tended to have pleiotropism.ZmCCT was found to modulate flowering time by photoperiodic pathway,as well as significantly enhance maize resistance to Gibberella stalk rot,indicateing that ZmCCT is a very important gene affecting maize production and safety.It is of great significance to compare the different molecular mechanism of ZmCCT in regulating different traits,which can promote the utilization of ZmCCT in breeding and production.In order to identify the difference of mechanism in different traits regulated by ZmCCT,this study firstly used the Pull-down combined MS method to find the interaction factors of ZmCCT based on the expression of ZmCCT protein in vitro.The result showed that 43 interacting proteins of ZmCCT were identified in leaves,which participated in pathway such as protein folding and refolding,photoperiodic response and regulation and transmembrane transport of substances.Meanwhile,the result also showed that 52 nteracting proteins of ZmCCT were identified in roots which participated in pathway related to protein binding,ion transmembrane transport and methyltransferase activity.After comparison,it was found that except for 2 proteins,the other identified proteins all specifically interacted in leaves or roots,which indicated that the cofactors of ZmCCT in leaves and roots were different,suggesting that the mechanisms of photoperiodic response in leaves and response to Fusarium graminearum in roots were also different.In leaves,ZmCCT might interact directly with Phytochrome A and act on other proteins such as 14-3-3 protein,so as to feel the photoperiod and regulate flowering.ZmCCT interacted with itself to form dimer,and the interaction between ZmCCT and 14-3-3 protein was confirmed by experiments.Considering the different interaction factors of ZmCCT in different tissues,in order to find the target genes regulated by ZmCCT in different tissues,we established an optimized Cell-free/DAP-seq system.In leaves,the DNA motifs most likely to be bound by ZmCCT were "CCGCCG" and "ATGGGCG",and there were 243 target genes directly regulated by ZmCTT,of which 12 genes contained both DNA motifs above.Simultaneously,in roots the DNA motifs most likely to be bound by ZmCCT were "GTGTAAAATG","AAAACAGCG"and "CAAATGG",and there were 102 target genes directly regulated by ZmCTT,of which 13 genes contained both DNA motifs above.The target genes screened above did not overlap in the two tissues,which indicated that ZmCCT can bind to different DNA motifs in leaves and roots,thereby regulating different target genes,further suggesting that the mechanisms of the response to photoperiod in leaves and the response to Fusarium graminearum in roots were different.This study laid a theoretical foundation for further study of the different mechanisms of ZmCCT in leaves and roots,and also provided theoretical guidance for the application of ZmCCT in molecular breeding.