Genome‑wide analysis of the maize CK1 family and functional characterization of ZmCK1-8 in modulating root architecture and nitrate‑responsive physiological traits under low‑nitrogen conditions

Abstract

Nitrogen deficiency is a major constraint of global maize production. Although CK1 family members have diverse regulatory functions in eukaryotes, the CK1 family in maize has not yet been systematically identified, and their roles in maize nitrogen metabolism remain poorly understood. In the current study, we sought to identify the ZmCK1 family members and investigate the physiological and molecular roles of ZmCK1s in maize growth under low-nitrogen conditions. We identified 20 maize ZmCK1 family members and characterized them through phylogenetic analysis, motif and domain prediction, gene structure analysis, promoter cis-element annotation, and nitrate-responsive transcriptome profiling. ZmCK1-8 , a nitrate-responsive gene, was selected for functional analysis. B73 and ZmCK1-8 mutants were hydroponically cultured under low-nitrogen conditions to assess root architecture, glutamine synthetase (GS), glutamate synthase (GOGAT), nitrate reductase (NR), and protein and nitrogen contents. The mutants exhibited enhanced root growth, suggesting that ZmCK1-8 plays a role in regulating root growth under low‑nitrogen condition and is associated with altered nitrogen‑related physiological traits. Transcriptomic analysis identified 256 and 660 genotype‑dependent nitrate‑responsive genes in ck1‑8‑1 and ck1‑8‑2 mutants, respectively, enriched in hormone signaling, stimulus responses, and transmembrane transporter activity. Loss of ZmCK1-8 was associated with upregulation of ZmGS1-5 , and altered nitrogen-related physiological traits. Yeast two-hybrid and BiFC assays confirmed that ZmCK1-8 physically interacts with the chromatin remodeler ZmCHB101. RT-qPCR analysis of ZmCHB101 RNAi line further suggested that ZmCK1-8 and ZmCHB101 share several downstream targets, including ZmGS1–5 , GOGAT , and ZmPTR11 . In contrast, other nitrate-responsive genes, such as ZmPTR12 , ZmNPF25 , and ZmNPF34 , showed distinct expression patterns between the two genetic backgrounds. Genome-wide analysis identified 20 ZmCK1 members, of which ZmCK1-8 was found to be associated with nitrate‑induced root growth. Loss of ZmCK1-8 function is associated with altered nitrogen-related physiological traits in roots, while ZmGS1-5 is identified as a candidate downstream responder. Furthermore, ZmCK1-8 physically interacts with ZmCHB101, though the mechanistic link between this interaction and target-gene regulation remains to be established. These results uncover a novel component of the maize nitrogen-signaling network and provide a candidate gene for further functional studies on low‑nitrogen adaptation in maize.