| Summary: | Maize is an important cereal crop worldwide and is sensitive to abiotic stresses in fluctuant environments that seriously affect its growth, yield, and quality. The small heat shock protein (<i>HSP20</i>) plays a crucial role in protecting plants from abiotic stress. However, little is known about <i>HSP20</i> in maize (<i>ZmHSP20</i>). In this study, 44 <i>ZmHSP20s</i> were identified, which were unequally distributed over 10 chromosomes, and 6 pairs of <i>ZmHSP20s</i> were tandemly presented. The gene structure of <i>ZmHSP20s</i> was highly conserved, with 95% (42) of the genes having no more than one intron. The analysis of the cis-element in <i>ZmHSP20s</i> promoter demonstrated large amounts of elements related to hormonal and abiotic stress responses, including abscisic acid (ABA), high temperature, and hypoxia. The ZmHSP20s protein had more than two conserved motifs that were predictably localized in the cytoplasm, nucleus, endoplasmic reticulum, peroxisome, mitochondria, and plasma. Phylogenetic analysis using HSP20s in <i>Arabidopsis</i>, rice, maize, and <i>Solanum tuberosum</i> indicated that ZmHSP20s were classified into 11 categories, of which each category had unique subcellular localization. Approximately 80% (35) of <i>ZmHSP20</i> were upregulated under heat stress at the maize seedling stage, whereas the opposite expression profiling of 10 genes under 37 and 48 °C was detected. A total of 20 genes were randomly selected to investigate their expression under treatments of ABA, gibberellin (GA), ethylene, low temperature, drought, and waterlogging, and the results displayed that more than half of these genes were downregulated while <i>ZmHSP20-3</i>, <i>ZmHSP20-7</i>, <i>ZmHSP20-24</i>, and <i>ZmHSP20-44</i> were upregulated under 1 h treatment of ethylene. A yeast-one-hybrid experiment was conducted to analyze the binding of four heat stress transcription factors (ZmHSFs) with eight of the <i>ZmHSP20s</i> promoter sequences, in which ZmHSF3, ZmHSF13, and ZmHSF17 can bind to most of these selected <i>ZmHSP20s</i> promoters. Our results provided a valuable resource for studying <i>HSP20s</i> function and offering candidates for genetic improvement under abiotic stress.
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