Heat shock factor ZmHsf17 positively regulates phosphatidic acid phosphohydrolase ZmPAH1 and enhances maize thermotolerance

Heat stress adversely impacts plant growth, development, and grain yield. Heat shock factors (Hsf), especially the HsfA2 subclass, play a pivotal role in the transcriptional regulation of genes in response to heat stress. In this study, the coding sequence of maize ZmHsf17 was cloned. ZmHsf17 contained conserved domains including a DNA binding domain, oligomerization domain, and transcriptional activation domain. The protein was nuclear localized and had transcription activation activity. Yeast two-hybrid and split luciferase complementation assays confirmed the interaction of ZmHsf17 with members of the maize HsfA2 subclass. Overexpression of ZmHsf17 in maize significantly increased chlorophyll content and net photosynthetic rate, and enhanced the stability of cellular membranes. Through integrative analysis of ChIP-seq and RNA-seq datasets, ZmPAH1, encoding phosphatidic acid phosphohydrolase of lipid metabolic pathways, was identified as a target gene of ZmHsf17. The promoter fragment of ZmPAH1 was bound by ZmHsf17 in protein–DNA interaction experiments in vivo and in vitro. Lipidomic data also indicated that the overexpression of ZmHsf17 increased levels of some critical membrane lipid components of maize leaves under heat stress. This research provides new insights into the role of the ZmHsf17–ZmPAH1 module in regulating thermotolerance in maize.

Methods
Plant materials and growth conditions

Stress treatments

Isolation and preparation of CDS

Cloning of ZmHsf17 CDS

The instructions for using the HEATSTER platform

Subcellular localization

Transcription activity and yeast two-hybrid assay

Split-luciferase complementation

Real-time quantitative reverse transcription PCR (qRT-PCR)

SDS-PAGE and Immunoblot analysis

ChIP-seq analysis

RNA-seq analysis

ChIP assay followed by qPCR (ChIP-qPCR)

Yeast one-hybrid assay (Y1H)

Electrophoretic mobility shift assay (EMSA)

Dual luciferase reporter assay

Lipidomics analysis by LC-MS/MS