Role of ZmCCT in drought tolerance of maize by transcriptome and DAP-seq experiments

Abiotic stressors are widespread in plants and can affect their growth, productivity, and quality. Previously, the maize transcription factor ZmCCT was been proved to be involved in the photoperiod response, delayed flowering and quantitative resistance to Gibberella stalk rot. In this study, we demonstrate that ZmCCT can regulate plant drought stress. ZmCCT physically interacted with ZmFra a 1, ZmWIPF2, and ZmAux/IAA8, which localised to the cytomembrane, cytoplasm, and nucleus, respectively, both in vitro and in vivo in a yeast two-hybrid screen in response to abiotic stress. Notably, ZmCCT recruits ZmWIPF2 to the nucleus, which has strong E3 self-ubiquitination activity dependent on its RING-H2 finger domain in vitro. When treated with higher indole-3-acetic acid (IAA)/abscisic acid (ABA) ratios, the height and root length of Y331-ΔTE increased, and this resulted in an increase in auxin and ABA tolerance. In vivo, ZmCCT promoted IAA biosynthesis in ZmCCT-overexpressed Arabidopsis. RNA-seq and DAP-seq analyses showed that ZmCCT can regulate the expression of ZmRD17, ZmAFP3, ZmPP2C and ZmARR16 under drought stress. Our finding provides a detailed overview of the molecular mechanism controlling ZmCCT functions and highlights that it HAS multiple roles in promoting stress tolerance. For maize RNA-seq analysis, pooled leaf tissues from three maize seedlings at three-leaf stage were collected from Y331-ΔTE (control, stalk rot-resistant) and Y331 (TE insertion caused non-functional ZmCCT) and plants before or after drought stress to conduct the RNA-seq analysis. Three biological replicates of RNA-seq were included for each condition. Further more, ZmAux/IAA8, a interacting partner of ZmCCT, was fused to Halotag and conducted DAP-seq (DNA Affinity Purification sequencing) to identify its target regions in maize genome. Two biological replicates of IP samples and one replicate of input samples were included for DAP-seq experiment.