# Identification of the ZmDUF966 Gene Family in Maize, Analysis of Its Expression Under Cold Stress, and Preliminary Investigation of the ZmDUF966-10 Regulatory Network

## TL;DR

This study identifies a maize gene family, ZmDUF966, and finds that ZmDUF966-10 is strongly activated under cold stress and may be regulated by microRNAs and ABA signaling.

## Contribution

The study identifies ZmDUF966-10 as a novel candidate gene involved in cold stress response in maize through multi-level regulatory mechanisms.

## Key findings

- ZmDUF966-10 is significantly up-regulated under cold stress and regulated by conserved miRNAs like zma-miR159.
- The ZmDUF966-10 protein interacts with an ABA/WDS-induced protein, suggesting a role in ABA-mediated stress signaling.
- The ZmDUF966 gene family is conserved in Poaceae species and contains stress-responsive promoter elements like LTR and ABRE.

## Abstract

Cold stress significantly limits maize growth and yield, particularly at the seedling stage. In this study, we systematically identified and characterized 10 members of the maize DUF966 gene family and analyzed their potential roles in cold stress response. Among them, ZmDUF966-10 was strongly induced under low-temperature conditions and may be regulated by conserved microRNAs and ABA-related signaling pathways. Preliminary interaction analysis suggests that this gene participates in stress-responsive regulatory networks. These findings provide a promising candidate gene for future functional studies and for improving cold tolerance in maize breeding programs.

Maize (Zea mays L.) originated in tropical and subtropical regions. During its growth and development, cold stress severely threatens seedling survival rates and final yield by inducing oxidative stress, compromising cell membrane integrity, and causing “physiological drought.” The Domain of Unknown Function 966 (DUF966) gene family comprises a class of regulatory factors containing conserved domains of undetermined function. Although they are considered to be extensively involved in plant growth, development, and stress response, their specific roles within the maize cold-tolerance regulatory network remain to be explored. In this study, 10 ZmDUF966 family members were identified via genome-wide analysis, and their phylogenetic relationships, gene structures, conserved motifs, chromosomal localizations, and cis-acting elements were systematically analyzed. The results indicate that the ZmDUF966 family is highly conserved among Poaceae species, and its promoters are enriched with stress-responsive elements such as LTR and ABRE. The core gene, ZmDUF966-10, was significantly up-regulated (approximately 35-fold at 48 h, p < 0.05) as validated by RT-qPCR under cold stress and is post-transcriptionally regulated by conserved miRNAs such as zma-miR159. Further yeast two-hybrid experiments revealed a preliminary physical interaction between the ZmDUF966-10 protein and an ABA/WDS-induced protein, suggesting its potential involvement in ABA-mediated stress signaling, though functional validation remains to be conducted. In conclusion, this study identifies ZmDUF966-10 as a promising candidate gene that responds to cold signals through multi-level regulatory networks, providing a valuable gene resource for further functional characterization and potential application in cold-tolerant maize improvement.