TaBI-1.1 is upregulated under heat stress
We analysed the RNA-seq data from heat-treated wheat to investigate the biological mechanism of the heat stress response. A highly upregulated gene, TaBI-1.1 (12.15-fold increase, TRIAE_CS42_U_TGACv1_644608_AA2140670), was identified from the RNA-seq data (Additional file 1: Table S1). To determine whether TaBI-1.1 was upregulated under heat stress, we monitored TaBI-1.1 expression using real time quantitative PCR (qRT-PCR). TaBI-1.1 mRNA accumulated during heat treatment, reaching a peak of ~ 6-fold at 8 h (Fig. 1a). We analysed the histological β-glucuronidase (GUS) activity via GUS staining of PBI::GUS transgenic Arabidopsis to investigate the spatial expression pattern of TaBI-1.1. Higher levels of GUS protein accumulation were observed in the mature leaves of heat-treated plants than in those of control plants (Fig. 1b and c). GUS mRNA levels were also obviously increased, as shown by qRT-PCR, confirming the upregulation of TaBI-1.1 under heat stress (Fig. 1d).
Constitutive expression of TaBI-1.1 in atbi1–2 fully rescues defective heat tolerance in atbi1–2 plants
BI-1 is known to be highly evolutionarily conserved. atbi1–2 mutants are hypersensitive to heat stress, and AtBI-1 overexpression can rescue this deficiency [17]. Based on the high upregulation of TaBI-1.1 under heat treatment, we surmised that TaBI-1.1 might play a similar role as AtBI-1 in the response to heat stress. To test this hypothesis, we generated transgenic lines that ectopically expressed TaBI-1.1 in the atbi1–2 background under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Two homozygous lines with relatively high TaBI-1.1 expression levels (35S::TaBI-1.1/atbi1–2#1 and 35S::TaBI-1.1/atbi1–2#2) were selected for further analysis. Eighteen-day-old atbi1–2 and Col-0 plants, as well as the two transgenic lines, were exposed to 45 °C for 6 h, and the survival rates were tested after 7 days. The survival rates of Col-0, 35S::TaBI-1.1/atbi1–2#1, and 35S::TaBI-1.1/atbi1–2#2 plants were not significantly different, and all the three genotypes exhibited significantly higher survival rates than atbi1–2. Compared with the survival rates of atbi1–2 and Col-0, the two transgenic lines fully rescued the deficiency of atbi1–2 under heat stress (Fig. 2a and b). Furthermore, we examined ion leakage in the different genotypes. Under normal conditions, no differences in relative conductivity were observed between any of the genotypes. However, under heat treatment, atbi1–2 showed a significantly higher relative conductivity than Col-0 and the two transgenic lines, and no differences in relative conductivity were detected between Col-0 and the two transgenic lines, which confirmed that TaBI-1.1 fully rescued the deficiency of atbi1–2 in response to heat stress, indicating the conserved function of BI-1 between wheat and Arabidopsis (Fig. 2c).
Hypocotyl elongation is known to be inhibited by heat stress [31]. To elucidate the role of BI-1 during hypocotyl elongation under heat stress, plants from all four genotypes were grown in the dark for 3 days and then exposed to 45 °C for 2 h. Hypocotyl elongation was tested after 3 days of recovery. Six-day-old plants grown in the dark were used as the control. In the control group, no differences in hypocotyl elongation were observed between atbi1–2 and Col-0, while the two transgenic lines displayed significantly shorter hypocotyls than Col-0. In contrast, atbi1–2 exhibited less hypocotyl elongation than the two transgenic lines and Col-0 under heat treatment (Fig. 2d and e). These results suggested that hypocotyl elongation in atbi1–2 is hypersensitive under heat stress and that TaBI-1.1 rescues the hypersensitive phenotype of atbi1–2.
Based on the conserved function of TaBI-1.1 and AtBI-1 under heat stress, as well as their high sequence conservation, we analysed the number of BI-1 members in some important species by screening the Ensembl plant database and constructed a phylogenetic tree of the BI-1 family (Additional file 2: Figure S1B). Aegilops tauschii, Arabidopsis thaliana, Brachypodium distachyon, Hordeum vulgare, and Oryza sativa each contained only one BI-1. Additionally, only three members were identified in Triticum aestivum and Zea mays (Additional file 2: Figure S1A). These results showed that several BI-1 members are present in plant species, revealing the pivotal role of BI-1 in plants.
TaBI-1.1 interacts with the TPR domain of TaFKBP62
To further explore the cellular mechanisms of TaBI-1.1 in heat tolerance, we performed yeast two-hybrid assays using TaBI-1.1 expressed from the pGBKT7 (BD) vector as the bait protein to screen a wheat cDNA library. One candidate interacting partner, the ROF1 homologue TaFKBP62, which shares 59.23% amino acid sequence identity with ROF1, was obtained. Both proteins contain three PPIase domains and one TPR domain and belong to the FKBP62 family. Their TPR domains share 70.94% identity, indicating that the TPR domain of FKBP62 is conserved between wheat and Arabidopsis.
Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays were used to explore the interaction between TaBI-1.1 and TaFKBP62. A BD vector containing TaBI-1.1 (BD-TaBI-1.1) and a pGADT7 (AD) vector containing TaFKBP62 (AD-TaFKBP62) were constructed for the yeast two-hybrid analysis. Four groups, BD-TaBI-1.1 + AD-TaFKBP62, BD-TaBI-1.1 + AD, BD + AD-TaFKBP62, and BD + AD, were co-transformed into yeast cells. Only yeast cells transformed with BD-TaBI-1.1 and AD-TaFKBP62 were able to grow on selective medium lacking Trp, Leu, His, and Ade (SD/−Trp-Leu-Ade-His). Conversely, co-transformants expressing BD-TaBI-1.1 + AD, BD + AD-TaFKBP62 or BD + AD did not grow on SD/−Trp-Leu-Ade-His medium. Similar results were obtained when we switched the TaBI-1.1 and TaFKBP62 vectors, indicating that TaBI-1.1 interacted with TaFKBP62 in yeast cells (Fig. 3a). This interaction was further demonstrated using BiFC assay. Four vector groups, TaBI-1.1–YFPN (N-terminal fragment of yellow fluorescent protein) and TaFKBP62–YFPC (C-terminal fragment of yellow fluorescent protein), TaBI-1.1–YFPN and YFPC, YFPN and TaFKBP62–YFPC, and YFPN and YFPC, were injected into Nicotiana benthamiana leaves. YFP fluorescence was only detected in leaf epidermal cells injected with TaBI-1.1–YFPN and TaFKBP62–YFPC (Fig. 3b), confirming the interaction between TaBI-1.1 and TaFKBP62.
To determine which region of TaFKBP62 interacted with TaBI-1.1, the TaFKBP62 sequence was divided to two fragments, TaFKBP62-PPIase (N-terminal and three PPIase domains) and TaFKBP62-TPR (TPR domain), according to the protein domains (Fig. 3c). The two fragments were cloned into an AD vector and co-transformed with BD-TaBI-1.1 into yeast cells. Both AD-TaFKBP62-PPIase + BD-TaBI-1.1 and AD-TaFKBP62-TPR + BD-TaBI-1.1 cells grew on SD/−Trp-Leu medium, whereas only transformants expressing AD-TaFKBP62-TPR and BD-TaBI-1.1 grew on SD/−Trp-Leu-His-Ade/X-α-gal medium (Fig. 3d). These results suggested that TaBI-1.1 specifically interacts with the TPR domain of TaFKBP62.
Given the conserved TPR domain between TaFKBP62 and ROF1, the sequence conservation between TaBI-1.1 and AtBI-1, and the interaction between TaFKBP62 and TaBI-1.1, we also tested the interaction between AtBI-1 and ROF1, as well as AtBI-1 and the TPR domain of ROF1. However, AtBI-1 did not interact with ROF1 or the TPR domain of ROF1 in yeast cells (Additional file 3: Figure S2).
TaFKBP62 co-localizes with TaBI-1.1 on the ER membrane and enhances the heat stress tolerance in Arabidopsis
We previously showed that TaBI-1.1 localizes to the ER membrane [32]. In view of the interaction between TaBI-1.1 and TaFKBP62, we constructed recombinant TaFKBP62-GFP and TaFKBP62-mRFP vectors to detect the subcellular localization of TaFKBP62 and to determine whether TaBI-1.1 co-localizes with TaFKBP62 at the ER membrane. Two groups, TaFKBP62-GFP + mRFP-HDEL (an ER marker) and TaBI-1.1-GFP + TaFKBP62-mRFP, were co-transformed into wheat protoplasts. The overlap coefficient for TaFKBP62-GFP and mRFP-HDEL fluorescence was 0.67, indicating that TaFKBP62 localized to the ER membrane in the wheat protoplasts (Fig. 4a). The overlap coefficient for TaBI-1.1-GFP and TaFKBP62-mRFP fluorescence was 0.63, suggesting that TaBI-1.1 co-localized with TaFKBP62 at the ER membrane (Fig. 4a). ROF1 localizes to the cytoplasm under normal conditions and translocates into the nucleus under heat treatment [30]. However, we did not observe the nuclear translocation of either TaFKBP62 or TaBI-1.1 under heat stress (Additional file 4: Figure S3).
To determine the possible role of TaFKBP62 under heat stress, we monitored TaFKBP62 expression patterns using qRT-PCR and found that TaFKBP62 obviously accumulated during heat treatment, reaching a peak point of ~ 5.3-fold at 0.5 h (Fig. 4b). TaFKBP62 upregulation was also detected in the RNA-seq data from heat-treated wheat (Additional file 5: Table S2). We generated transgenic Arabidopsis plants that constitutively expressed TaFKBP62 under the control of the CaMV 35S promoter. Three homozygous lines, 35S::TaFKBP62–1 35S::TaFKBP62–2 and 35S::TaFKBP62–3, were selected for further analysis. Eighteen-day-old Col-0 and the three transgenic lines were exposed to 45 °C for 6 h, and survival rates were tested after 7 days. The survival rates of the three transgenic lines were significantly higher than those of Col-0 (Fig. 4c and d). Ion-leakage assays showed that the three transgenic lines exhibited significantly lower relative conductivity than Col-0 under heat stress, indicating that TaFKBP62 enhanced heat stress tolerance in Arabidopsis (Fig. 4e).
TaBI-1.1 is conserved with AtBI-1 in regulating heat-responsive gene expression
We conducted an RNA-seq analysis of atbi-1 and Col-0 under heat treatment to further investigate the mechanism of BI-1 in response to heat stress. Thirty-five upregulated genes and 80 downregulated genes were identified (Additional file 6: Figure S4). The gene identity (ID) numbers and fold-changes are shown in Additional file 5: Table S2. Most of the HSPs were positively regulated under heat stress. Therefore, we analysed the downregulated genes in atbi-1 versus (vs) Col-0. The top 30 enriched Gene Ontology (GO) terms and the top 20 enriched Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways among the downregulated genes are shown in Fig. 5a and b. Only the term chaperone activity was significant among the 30 enriched GO terms. Of the top 20 enriched pathways, the greater enrichment factor represented a higher degree of enrichment. The enriched pathway protein processing in the ER had the highest enrichment factor, indicating that most of the downregulated genes were involved in the protein processing pathway in the ER. Eight differentially expressed genes, HSFA2, HSFB1, ROF1, HSP17.4B, HSP17.6A, HSP17.8, HSP70B, and HSP90.1, were selected to generate a histogram for visual analysis based on their fragments per kilobase of transcript per million mapped reads (FPKM) in atbi-1 and Col-0. The FPKM of these genes was significantly lower in atbi-1 than in Col-0 (Fig. 5c), revealing that atbi1–2 deficiency might be caused by the downregulation of these genes under heat stress and that the loss of AtBI-1 function might affect protein processing and chaperone activity under heat stress.
To verify the accuracy of the RNA-seq analysis and investigate whether TaBI-1.1 can rescue the downregulation of these genes in atbi1–2, qRT-PCR analyses were performed using the aforementioned eight genes. Under normal conditions (0 h), no significant differences in gene expression levels were detected among the three genotypes. After heat treatment for 1 h, all eight genes showed obvious upregulation, indicating that they were heat-responsive genes. The expression levels of these genes were significantly lower in atbi1–2 than in Col-0. In contrast, no significant differences in gene expression were observed between Col-0 and 35S::TaBI-1.1/atbi1–2 (Fig. 6). These results suggested that TaBI-1.1 fully rescued the downregulation of these heat-responsive genes in atbi1–2.