In this study, we were dedicated to investigating the effect caused by heat stress on wheat flag leaves. Metabolome and transcriptome analysis were introduced to identify some key biological processes. As a result, 182 and 214 metabolites were significantly changed at the anthesis and post-anthesis stages, respectively; most of them were lipids, amino acids and derivatives, phenolic acids, and alkaloids. Aminoacyl-tRNA biosynthesis was the most significantly enriched pathway by metabolites at both two stages, each of which included 13 types of amino acid, and 12 of them were shared and up-regulated. Therefore, we further measured 22 kinds of amino acid content in ten different wheat genotypes at the post-anthesis stage. Based on the average content of each amino acid, 17 kinds of them were significantly increased after heat stress, and 4 types were significantly decreased. Both the metabolism analysis and the transcriptome analysis had a higher number of significantly changed metabolites or differential expressed genes at the post-anthesis stage, which indicated that the post-anthesis stage is more sensitive to heat stress, with 21,361 and 17,130 differential expressed genes, respectively. Two pathways, protein processing in endoplasmic reticulum and ABC transporters, were significantly enriched at two stages. The differential expressed genes in processing in endoplasmic reticulum pathway mainly encoded various types of molecular chaperones; among them, the HSP20 family was the most predominant and intensively up-regulated. The ABC transporter gene family is another pathway that is deeply involved in heat-stress response, which could be classified into five subfamilies; among them, subfamilies B and G were the most active. In summary, this study revealed the heat response pattern of amino acids, HSPs, and ABC transporter which may play a vital role during the wheat reproductive stage.

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.

Red-skinned pears are favored by people for their attractive appearance and abundance of anthocyanins. However, the molecular basis of anthocyanin biosynthesis in red pears remains elusive. Here, a comprehensive transcriptome analysis was conducted to explore the potential regulatory mechanism of anthocyanin biosynthesis in ‘Red Zaosu’ pear (Pyrus pyrifolia × Pyrus communis). Gene co-expression analysis and transcription factor mining identified 263 transcription factors, which accounted for 6.59% of the total number of transcription factors in the pear genome in two gene modules that are highly correlated with anthocyanin biosynthesis. Clustering, gene network modeling with STRING-DB, and local motif enrichment analysis (CentriMo) analysis suggested that PpPIF8 may play a role in anthocyanin biosynthesis. Furthermore, eight PIFs were identified in the pear genome, of which only PpPIF8 was rapidly induced by light. Functional studies showed that PpPIF8 localizes in the nucleus and is preferentially expressed in the tissue of higher levels of anthocyanin. The overexpression of PpPIF8 in pear peel and pear calli promotes anthocyanin biosynthesis and upregulates the expression of anthocyanin biosynthesis genes. Yeast-one hybrid and transgenic analyses indicated that PpPIF8 binds to the PpCHS promoter to induce PpCHS expression. The positive effect of PpPIF8 on anthocyanin biosynthesis is different from previously identified negative regulators of PyPIF5 and MdPIF7 in pear and apple. Taken together, our data not only provide a comprehensive view of transcription events during the coloration of pear peel, but also resolved the regulatory role of PpPIF8 in the anthocyanin biosynthesis pathway.

‘Huangguan’ pear (Pyrus bretschneideri Rehd) fruit is susceptible to cold, characterized by developing peel browning spots (PBS) during cold storage. Additionally, ethylene pretreatment reduces chilling injury (CI) and inhibits PBS occurrence, but the mechanism of CI remains unclear. Here, we deciphered the dynamic transcriptional changes during the PBS occurrence with and without ethylene pretreatment via time-series transcriptome. We found that ethylene suppressed the cold-signaling gene expression, thereby decreasing the cold sensitivity of the ‘Huangguan’ fruit. Moreover, the “Yellow” module closely correlated with PBS occurrence was identified via weighted gene co-expression network analysis (WGCNA), and this module was related to plant defense via Gene Ontology (GO) enrichment analysis. Local motif enrichment analysis suggested that the “Yellow” module genes were regulated by ERF and WRKY transcription factors. Functional studies demonstrated that PbWRKY31 has a conserved WRKY domain, lacks transactivation activity, and localizes in the nucleus. PbWRKY31-overexpressed Arabidopsis were hypersensitive to cold, with higher expression levels of cold signaling and defense genes, suggesting that PbWRKY31 participates in regulating plant cold sensitivity. Collectively, our findings provide a comprehensive transcriptional overview of PBS occurrence and elucidate the molecular mechanism by which ethylene reduces the cold sensitivity of ‘Huangguan’ fruit as well as the potential role of PbWRKY31 in this process.

The dramatically high temperatures triggered by global climate change threaten maize growth and yield. In recent years, increasing attention has focused on the impacts of heat injury on maize. However, the molecular mechanisms behind maize’s adaptation to heat stress remain largely unexplored. To uncover how plants protect themselves from heat stress, we performed a phosphoproteomic analysis on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. A total of 1594 phosphopeptides ascribed to 875 proteins were identified. A functional enrichment analysis of the proteins and phosphoproteins revealed that the early thermal responses of maize were associated with translational and post-translational modifications, protein turnover, and chaperone binding in the MAPK pathway. A motif analysis also yielded a significant number of potential MAPK substrates. The functional characterization of the phosphoproteins and pathways identified here will provide new insights for improving crop thermal tolerance.