Abstract：Abstract Heat shock transcription factors (Hsfs) are the central regulators of the heat shock responsive genes that encode Hsps, and can specifically bind to heat shock elements (HSE) in the upstream promoter region of heat shock protein genes to realize the regulation to these genes. There are at least 30 Hsf members in maize(Zea mays) Hsf family which includes 18 members of class A, among which there are 4 Hsfs of subclass A2. In our previous work, the structure, characteristics and regulating roles in thermotolerance and drought-stress tolerance of ZmHsf06 have been investigated. In present study, ZmHsf04 was cloned from maize young leaves treated by heat shock at 42 ℃ for 1 h using homologous cloning methods. The coding sequence (CDS) of ZmHsf04 was 1 074 bp encoding a protein of 357 amino acids. Protein sequence of ZmHsf04 contained DNA-binding domain (DBD) and other functional domains such as a nuclear localization signal(NLS)of KRKELEDTISKKRRR, NES(LAQQLGYL) and AHA(LKMFESGVLN). Homologous analysis showed that ZmHsf04 protein sequence shared 90%, 86% and 78% identities with the hypothetical protein SORBIDRAFT_01g021490(XP_002467215.1) of Sorghum bicolor, SiHsfA2c-like(XP_012698415.1) of Setaria italic and OsHsfA2c-like(XP_006661780.2) of Oryza brachyantha, respectively. qRT-PCR results showed that ZmHsf04 was expressed in multiple tissues and organs of maize. Compared with young leaf, young root, stem, functional leave, immature embryo and ear, transcriptional expression level of ZmHsf04 was the highest in pollens with the value of 16 times of the control. ZmHsf04 expression was up-regulated significantly by both 42 ℃ heat shock and abscisic acid (ABA), the highest value reached 340 times of the control, and the peak values appeared at 30 min and 24 h, respectively. Meanwhile, ZmHsf04 expression was up-regulated by both salicylic acid(SA) and H2O2, and the highest value was 12 times of the control, and was significantly lower than that treated by HS or ABA. If pretreated with SA or H2O2 and then heat shock (HS), the ZmHsf04 expression was similar to that treated with HS. Through transient reporter assay with onion (Allium cepa) epidermal cells, it was found that ZmHsf04 was located in nuclei. ZmHsf04 protein could be induced by Gal in yeast (Saccharomyces cerevisiae), and yeast overexpressing pYES2-ZmHsf04 showed stronger growth potential than the controls expressing pYES2 after HS, though yeast growth potential was decreased by HS. The results revealed that ZmHsf04 perhaps played a key role in regulating pollen development and the response to heat stress. These results will provide theoretical basis for analysis biological functions and regulating mechanism of ZmHsf04 further.
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