Heat shock transcription factor (Hsf) plays a transcriptional regulatory role in plants during heat stress and other abiotic stresses. 31 non-redundant ZmHsf genes from maize were identified and clustered in the reference genome sequenced by Single Molecule Real Time (SMRT). The amino acid length, chromosome location, and presence of functional domains and motifs of all ZmHsfs sequences were analyzed and determined. Phylogenetics and collinearity analyses reveal gene duplication events in Hsf family and collinearity blocks shared by maize, rice and sorghum. The results of RNA-Seq analysis of anthesis and post-anthesis periods in maize show different expression patterns of ZmHsf family members. Specially, ZmHsf26 of A2 subclass and ZmHsf23 of A6 subclass were distinctly up-regulated after heat shock (HS) at post-anthesis stage. Nanopore transcriptome sequencing of maize seedlings showed that alternative splicing (AS) events occur in ZmHsf04 and ZmHsf17 which belong to subclass A2 after heat shock. Through sequence alignment, semi-quantitative and quantitative RT-PCR, we found that intron retention events occur in response to heat shock, and newly splice isoforms, ZmHsf04-II and ZmHsf17-II, were transcribed. Both new isoforms contain several premature termination codons in their introns which may lead to early termination of translation. The ZmHsf04 expression was highly increased than that of ZmHsf17, and the up-regulation of ZmHsf04-I transcription level were significantly higher than that of ZmHsf04-II after HS.

Heat stress (HS) adversely impacts plant growth, development and grain yield. Heat shock factors (Hsf), especially HsfA2 subclass, play a pivotal role in the transcriptional regulation of genes in response to HS. In this study, the coding sequence of maize ZmHsf17 was cloned. ZmHsf17 contains conserved domains: DNA binding, oligomerization and transcriptional activation. The protein was nuclear localized and had transcription activation activity. Yeast two hybrid and split luciferase complementary assays confirmed the interaction of ZmHsf17 with members of the maize HsfA2 subclass. Overexpression of ZmHsf17 in maize significantly increased chlorophyll content and net photosynthesis rate of maize leaves, 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 indicates that the overexpression of ZmHsf17 increased levels of some critical membrane lipid components of maize leaves under HS. This research provides new insights into the role of the ZmHsf17-ZmPAH1 module in regulating thermotolerance in maize.

To study the toxic mechanisms of lead (Pb) in plants, the effects of Pb on the morphology and structure of the nucleolus in root tip meristematic cells of Allium cepa var. agrogarum L. were investigated. Fluorescence labeling, silver-stained indirect immunofluorescent microscopy and western blotting were used. Fluorescence labeling showed that Pb ions were localized in the meristematic cells and the uptake and accumulation of Pb increased with treatment time. At low concentrations of Pb (1–10 μM) there were persistent nucleoli in some cells during mitosis, and at high concentration (100 μM) many of the nucleolar organizing regions were localized on sticky chromosomes in metaphase and anaphase cells. Pb induced the release of particles containing argyrophilic proteins to be released from the nucleus into the cytoplasm. These proteins contained nucleophosmin and nucleolin. Pb also caused the extrusion of fibrillarin from the nucleus into the cytoplasm. Western blotting demonstrated the increased expression of these three major nucleolar proteins under Pb stress.

High temperature has severely affected plant growth and development, resulting in reduced production of crops worldwide, especially wheat. Alternative splicing (AS), a crucial post-transcriptional regulatory mechanism, is involved in the growth and development of eukaryotes and the adaptation to environmental changes. Previous transcriptome data suggested that heat shock transcription factor (Hsf) TaHsfA2-7 may form different transcripts by AS. However, it remains unclear whether this post-transcriptional regulatory mechanism of TaHsfA2-7 is related to thermotolerance in wheat (Triticum aestivum). Here, we identified a novel splice variant, TaHsfA2-7-AS, which was induced by high temperature and played a positive role in thermotolerance regulation in wheat. Moreover, TaHsfA2-7-AS is predicted to encode a small truncated TaHsfA2-7 isoform, retaining only part of the DNA-binding domain (DBD). TaHsfA2-7-AS is constitutively expressed in various tissues of wheat. Notably, the expression level of TaHsfA2-7-AS is significantly up-regulated by heat shock (HS) during flowering and grain-filling stages in wheat. Further studies showed that TaHsfA2-7-AS was localized in the nucleus but lacked transcriptional activation activity. Ectopic expression of TaHsfA2-7-AS in yeast exhibited improved thermotolerance. Compared to non-transgenic plants, overexpression of TaHsfA2-7-AS in Arabidopsis results in enhanced tolerance to heat stress. Simultaneously, we also found that TaHsfA1 is directly involved in the transcriptional regulation of TaHsfA2-7 and TaHsfA2-7-AS. In summary, our findings demonstrate the function of TaHsfA2-7-AS splicing variant in response to heat stress and establish a link between regulatory mechanisms of AS and the improvement of thermotolerance in wheat.

Drought stress can seriously affect the yield and quality of wheat (Triticum aestivum). So far, although few wheat heat shock transcription factors (Hsfs) have been found to be involved in the stress response, the biological functions of them, especially the members of the HsfC (heat shock transcription factor C) subclass, remain largely unknown. Here, we identified a class C encoding gene, TaHsfC3-4, based on our previous omics data and analyzed its biological function in transgenic plants. TaHsfC3-4 encodes a protein containing 274 amino acids and shows the basic characteristics of the HsfC class. Gene expression profiles revealed that TaHsfC3-4 was constitutively expressed in many tissues of wheat and was induced during seed maturation. TaHsfC3-4 could be upregulated by PEG and abscisic acid (ABA), suggesting that this Hsf may be involved in the regulation pathway depending on ABA in drought resistance. Further results represented that TaHsfC3-4 was localized in the nucleus but had no transcriptional activation activity. Notably, overexpression of TaHsfC3-4 in Arabidopsis thaliana pyr1pyl1pyl2pyl4 (pyr1pyl124) quadruple mutant plants complemented the ABA-hyposensitive phenotypes of the quadruple mutant including cotyledon greening, root elongation, seedling growth, and increased tolerance to drought, indicating positive roles of TaHsfC3-4 in the ABA signaling pathway and drought tolerance. Furthermore, we identified TaHsfA2-11 as a TaHsfC3-4-interacting protein by yeast two-hybrid (Y2H) screening. The experimental data show that TaHsfC3-4 can indeed interact with TaHsfA2-11 in vitro and in vivo. Moreover, transgenic Arabidopsis TaHsfA2-11 overexpression lines exhibited enhanced drought tolerance, too. In summary, our study confirmed the role of TaHsfC3-4 in response to drought stress and provided a target locus for marker-assisted selection breeding to improve drought tolerance in wheat.