Plant heat shock transcription factors (HSFs) are involved in heat and other abiotic stress responses. However, their functions in salt tolerance are little known. In this study, we characterized the function of a HSF from Arabidopsis, AtHSFA7b, in salt tolerance. AtHSFA7b is a nuclear protein with transactivation activity. ChIP-seq combined with an RNA-seq assay indicated that AtHSFA7b preferentially binds to a novel cis-acting element, termed the E-box-like motif, to regulate gene expression; it also binds to the heat shock element motif. Under salt conditions, AtHSFA7b regulates its target genes to mediate serial physiological changes, including maintaining cellular ion homeostasis, reducing water loss rate, decreasing reactive oxygen species accumulation, and adjusting osmotic potential, which ultimately leads to improved salt tolerance. Additionally, most cellulose synthase-like (CSL) and cellulose synthase (CESA) family genes were inhibited by AtHSFA7b; some of them were randomly selected for salt tolerance characterization, and they were mainly found to negatively modulate salt tolerance. By contrast, some transcription factors (TFs) were induced by AtHSFA7b; among them, we randomly identified six TFs that positively regulate salt tolerance. Thus, AtHSFA7b serves as a transactivator that positively mediates salinity tolerance mainly through binding to the E-box-like motif to regulate gene expression.

Gene expression profiles are powerful tools for investigating mechanisms of plant stress tolerance. Betula platyphylla (birch) is a widely distributed tree, but its drought-tolerance mechanism has been little studied. Using RNA-Seq, we identified 2917 birch genes involved in its response to drought stress. These drought-responsive genes include the late embryogenesis abundant (LEA) family, heat shock protein (HSP) family, water shortage-related and ROS-scavenging proteins, and many transcription factors (TFs). Among the drought-induced TFs, the ethylene responsive factor (ERF) and myeloblastosis oncogene (MYB) families were the most abundant. BpERF2 and BpMYB102, which were strongly induced by drought and had high transcription levels, were selected to study their regulatory networks. BpERF2 and BpMYB102 both played roles in enhancing drought tolerance in birch. Chromatin immunoprecipitation combined with qRT-PCR indicated that BpERF2 regulated genes such as those in the LEA and HSP families, while BpMYB102 regulated genes such as Pathogenesis-related Protein 1 (PRP1) and 4-Coumarate:Coenzyme A Ligase 10 (4CL10). Multiple genes were regulated by both BpERF2 and BpMYB102. We further characterized the function of some of these genes, and the genes that encode Root Primordium Defective 1 (RPD1), PRP1, 4CL10, LEA1, SOD5, and HSPs were found to be involved in drought tolerance. Therefore, our results suggest that BpERF2 and BpMYB102 serve as transcription factors that regulate a series of drought-tolerance genes in B. platyphylla to improve drought tolerance.

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DNA binding proteins carry out important and diverse functions in the cell, including gene regulation, but identifying these proteins is technically challenging. In the present study, we developed a technique to capture DNA-associated proteins called reverse chromatin immunoprecipitation (R-ChIP). This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin, and the DNA-associated proteins are then identified using mass spectrometry. Using R-ChIP, we identified 439 proteins that potentially bind to the promoter of the Arabidopsis thaliana gene AtCAT3 (AT1G20620). According to functional annotation, we randomly selected 5 transcription factors from these candidates, including bZIP1664, TEM1, bHLH106, BTF3, and HAT1, to verify whether they in fact bind to the AtCAT3 promoter. The binding of these 5 transcription factors was confirmed using chromatin immunoprecipitation quantitative real-time PCR and electrophoretic mobility shift assays. In addition, we improved the R-ChIP method using plants in which the DNA of interest had been transiently introduced, which does not require the T-DNA integration, and showed that this substantially improved the protein capture efficiency. These results together demonstrate that R-ChIP has a wide application to characterize chromatin composition and isolate upstream regulators of a specific gene.