Wheat Bax Inhibitor-1 interacts with TaFKBP62 and mediates response to heat stress

Abstract

Background: Heat stress is a severe environmental stress that affects plant growth and reduces yield. Bax inhibitor-1 (BI-1) is a cytoprotective protein that is involved in the response to biotic and abiotic stresses. The Arabidopsis (Arabidopsis thaliana) BI-1 mutants atbi1-1 and atbi1-2 are hypersensitive to heat stress, and AtBI-1 overexpression rescues thermotolerance deficiency in atbi1 plants. Nevertheless, the mechanism of BI-1 in plant thermotolerance is still unclear.

Results: We identified a wheat (Triticum aestivum L.) BI-1 gene, TaBI-1.1, which was highly upregulated in an RNA sequencing (RNA-seq) analysis of heat-treated wheat. The upregulation of TaBI-1.1 under heat stress was further demonstrated by real time quantitative PCR (qRT-PCR) and β-glucuronidase (GUS) staining. Compared with the wild type Col-0, the atbi1-2 mutant is hypersensitive to heat stress, and constitutive expression of TaBI-1.1 in atbi1-2 (35S::TaBI-1.1/ atbi1-2) rescued the deficiency of atbi1-2 under heat stress. Furthermore, we identified TaFKBP62 as a TaBI-1.1-interacting protein that co-localized with TaBI-1.1 on the endoplasmic reticulum (ER) membrane and enhanced heat stress tolerance. Additionally, HSFA2, HSFB1, ROF1, HSP17.4B, HSP17.6A, HSP17.8, HSP70B, and HSP90.1 expression levels were suppressed in atbi1-2 plants under heat stress. In contrast, 35S::TaBI-1.1/atbi1-2 relieved the inhibitory effect of AtBI-1 loss of function.

Conclusions: TaBI-1.1 interacted with TaFKBP62 and co-localized with TaFKBP62 on the ER membrane. Both TaBI-1.1 and AtBI-1 regulated the expression of heat-responsive genes and were conserved in plant thermotolerance.

Keywords: Bax inhibitor-1; Heat stress; Heat-responsive genes; RNA-seq; TaFKBP62.

Fig. 1

Relative and spatial expression patterns of TaBI-1.1. a Expression profile of TaBI-1.1 under heat treatment for 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Wheat Actin was used as a reference. The vertical coordinates are the fold-changes, and the horizontal ordinates are the different time periods. b and c GUS activity of PBI::GUS transgenic Arabidopsis under normal conditions (b) and heat treatment (c) was detected via GUS histochemical staining. Seedlings grown under normal conditions were used as controls. d GUS expression levels in the PBI:GUS transgenic lines were monitored by qRT-PCR. Actin2 was used as a reference. The results are shown as the means±standard deviation (SD) of three biological replicates. Error bars indicate the SD. Asterisks (**) indicate the significant differences (P < 0.01) compared with Col-0 (Student’s t-test)

Fig. 2

Constitutive TaBI-1.1 expression in atbi1–2 rescues the deficiency of atbi1–2 in heat tolerance. a The phenotypes of atbi1–2, Col-0, 35S::TaBI-1.1/atbi1–2#1, and 35S::TaBI-1.1/atbi1–2#2 under heat stress. b The survival rates of atbi1–2, Col-0, 35S::TaBI-1.1/atbi1–2#1, and 35S::TaBI-1.1/atbi1–2#2 under heat stress. The results are shown as the means±SD of three biological replicates. Error bars indicate the SD. c The relative conductivity of atbi1–2, Col-0, 35S::TaBI-1.1/atbi1–2#1, and 35S::TaBI-1.1/atbi1–2#2 after heat treatment. The results are shown as the means±SD of six biological replicates. Error bars indicate the SD. d Hypocotyl elongation in atbi1–2, Col-0, 35S::TaBI-1.1/atbi1–2#1, and 35S::TaBI-1.1/atbi1–2#2 after heat treatment. e Statistical analysis of hypocotyl elongation in atbi1–2, Col-0, 35S::TaBI-1.1/atbi1–2#1, and 35S::TaBI-1.1/atbi1–2#2 after heat treatment. The results are shown as the means±SD of 36 biological replicates. Error bars indicate the SD. All the asterisks in the figure (* and **) indicate significant differences (P < 0.05 and P < 0.01, respectively) compared with Col-0 (Student’s t-test)

Fig. 3

The interaction between TaBI-1.1 and TaFKBP62. a The interaction between TaBI-1.1 and TaFKBP62 measured using yeast two-hybrid assays. b The interaction between TaBI-1.1 and TaFKBP62 measured via BiFC assays in Nicotiana benthamiana epidermal cells. Bars = 50 μm. c The two fragments, TaFKBP62-PPIase and TaFKBP62-TPR, corresponding to the PPIase and TPR domain, respectively, of TaFKBP62. d Interactions between TaBI-1.1 and the two TaFKBP62 fragments detected via yeast two-hybrid assay

Fig. 4

The co-localization between TaBI-1.1 and TaFKBP62 and TaFKBP62 expression patterns under stress treatments. a The subcellular localization of TaFKBP62 and the co-localization between TaBI-1.1 and TaFKBP62 in wheat protoplasts. The left panel shows the fluorescence of GFP, mRFP and the merge. Bars = 10 μm. The right panel shows the co-localization levels calculated from the overlap coefficients obtained from at least ten individual protoplasts. b TaFKBP62 expression patterns after heat treatment for 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Wheat Actin was used as a reference. The vertical coordinates represent the fold-changes, and the horizontal ordinates represent the different time periods. The results are shown as the means±SD of three biological replicates. Error bars indicate the SD. c The phenotypes of Col-0, 35S::TaFKBP62–1, 35S::TaFKBP62–2, and 35S::TaFKBP62–3 after heat stress. d The survival rates of Col-0, 35S::TaFKBP62–1, 35S::TaFKBP62–2, and 35S::TaFKBP62–3 under heat stress. The results are shown as the means±SD of three biological replicates. Error bars indicate the SD. e The relative conductivity of Col-0, 35S::TaFKBP62–1, 35S::TaFKBP62–2, and 35S::TaFKBP62–3 after heat treatment. The results are shown as the means±SD of six biological replicates. Error bars indicate the SD

Fig. 5

GO term enrichment and KEGG enrichment analyses were conducted using the RNA-seq data. a GO term enrichment analysis of the downregulated genes. The vertical coordinates are the enriched GO terms, and the horizontal coordinates are the numbers of the downregulated genes in these GO terms. The green columns represent the biological process GO terms; the purple columns represent the cellular component GO terms; the orange columns represent the molecular function GO terms. “*” indicates a significantly enriched GO term. b KEGG enrichment analysis of the downregulated genes. The vertical coordinates are the enriched pathways, and the horizontal coordinates are the rich factors. The size of each point represents the number of downregulated genes in the pathway, and the colour of the point represents the q-value. c FPKM values of the eight downregulated genes. Asterisks (*) indicate significant differences (padj< 0.005) compared with Col-0

Fig. 6

The expression levels of eight genes in atbi1–2, Col-0 and 35S::TaBI-1.1/atbi1–2 detected by qRT-PCR after heat treatment. Gene expression levels in Col-0 at 0 h were set to “1.” The results are shown as the means±SD of three biological replicates. Error bars indicate the SD. Asterisks (* and **) indicate significant differences (P < 0.05 and P < 0.01, respectively) compared with Col-0 (Student’s t-test)