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. 2023 Mar 3;13(3):687.
doi: 10.3390/life13030687.

Characterization of AtBAG2 as a Novel Molecular Chaperone

Chang Ho Kang  1 , Jae Hyeok Lee  1 , Yeon-Ju Kim  2 , Cha Young Kim  3 , Soo In Lee  4 , Jong Chan Hong  1 , Chae Oh Lim  1
Affiliations

Characterization of AtBAG2 as a Novel Molecular Chaperone

Chang Ho Kang et al. Life (Basel). .

Abstract

Bcl-2-associated anthanogene (BAG) family proteins regulate plant defense against biotic and abiotic stresses; however, the function and precise mechanism of action of each individual BAG protein are not yet clear. In this study, we investigated the biochemical and molecular functions of the Arabidopsis thaliana BAG2 (AtBAG2) protein, and elucidated its physiological role under stress conditions using mutant plants and transgenic yeast strains. The T-DNA insertion atbag2 mutant plants were highly susceptible to heat shock, whereas transgenic yeast strains ectopically expressing AtBAG2 exhibited outstanding thermotolerance. Moreover, a biochemical analysis of GST-fused recombinant proteins produced in bacteria revealed that AtBAG2 exhibits molecular chaperone activity, which could be attributed to its BAG domain. The relevance of the molecular chaperone function of AtBAG2 to the cellular heat stress response was confirmed using yeast transformants, and the experimental results showed that overexpression of the AtBAG2 sequence encoding only the BAG domain was sufficient to impart thermotolerance. Overall, these results suggest that the BAG domain-dependent molecular chaperone activity of AtBAG2 is indispensable for the heat stress response of Arabidopsis. This is the first report demonstrating the role of AtBAG2 as a sole molecular chaperone in Arabidopsis.

Keywords: BAG (Bcl-2-associated anthanogene) family proteins; abiotic stress; molecular chaperone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
AtBAG2 regulates heat stress tolerance in Arabidopsis. (A) Schematic representation of the predicted gene structure of AtBAG2. Black elbow arrows represent the start codon (ATG). Filled boxes and horizontal black lines indicate exons and introns, respectively. Empty boxes at either end of the gene represent 5′ and 3′ untranslated regions (UTRs). A black arrowhead indicates the T-DNA position in atbag2. Black arrows represent the binding sites of primers used for genotyping. (B,C) Confirmation of homozygous atbag2 mutant lines by genomic DNA-based PCR (B) and sqRT-PCR (C). (D) Photographs of WT (Col-0) and atbag2 seedlings grown at optimal temperature (22 °C) for 14 days (control) or subjected to heat stress conditions (7-day-old seedlings treated with heat shock [44 °C] for 30 min, and then grown at 22 °C for 7 days). (E,F) Comparison of survival rate (E) and ion leakage (F) between 7-day-old WT (Col-0) and atbag2 seedlings subjected to the heat shock treatment, as described in (D). In (E), the survival rates of heat-stressed Col-0 and atbag2 seedlings (heat shock) were compared with those of unstressed plants (control). Data represent the mean ± standard deviation (SD) of at least three independent experiments. To measure the ion leakage of Col-0 (-●-) and atbag2 (-○-) seedlings shown in (F), samples were collected at the indicated times and submerged in deionized water for 1 day. The conductivity of at least 10 seedlings was measured before autoclaving (initial conductivity) and after autoclaving (final conductivity). Data represent the mean ± SD of at least three independent experiments.
Figure 2
Figure 2
Ectopic expression of AtBAG2 enhances the thermotolerance of yeast cells. (A) Effect of heat stress on yeast cell viability. Yeast cells transformed with pYES2-G-AtBAG2 (G-AtBAG2) or pYES2-G (G) were grown in YPD media supplemented with 2% galactose (Gal). The transformed cells (5 × 107 cells/mL) were incubated at 27 °C or 55 °C and sampled at the indicated time points to count the number of viable cells. The cell survival rate (%) of each transformant was calculated as the ratio of the viable cell count at a given time point to the viable cell count at the 0 min time point. Data represent the mean ± SD of at least three independent experiments. (B) TB exclusion assay. Samples of G-AtBAG2 and G transformants incubated at 55 °C for 1 h in (A) were visualized by fluorescence microscopy after staining with TB. White and black arrowheads indicate TB-negative and -positive cells, respectively. Scale bars, 10 μm. Data represent the mean ± SD of at least three independent experiments.
Figure 3
Figure 3
AtBAG2 acts as a molecular chaperone. (A) Results of bis-ANS-binding assay used to investigate the effect of heat shock on the hydrophobic domains of GST alone (G), GST-AtBAG2 fusion (G-AtBAG2), and G-Ypt1p fusion (G-Ypt1p; control). The fluorescence spectra of 10 μM bis-ANS (-○-) and 10 μM bis-ANS plus 30 μM G-AtBAG2 (-▲-), G-Ypt1p (-■-), or G (-●-) were measured at 380 nm (excitation wavelength) and 400–600 nm (emission wavelengths). (B–D) Molecular chaperone activity assay of AtBAG2 using CS (B), MDH (C), and insulin (D). Light scattering was monitored at 340 nm. In (B), 1 μM CS was incubated either alone (-○-) or with 2 μM G (-●-), G-Ypt1p (-■-), or G-AtBAG2 (-▲-) in 50 mM HEPES buffer (pH 7.0) in a spectrophotometer cell at 43 °C. In (C), 1.67 μM MDH was incubated alone (-○-), with 0.84 μM (-▲-), 1.67 μM (-▲-), or 3.34 μM (-▲-) G-AtBAG2, or with 3.34 μM G (-●-) in 50 mM HEPES buffer (pH 7.0) in a spectrophotometer cell at 45 °C. In (D), 1 μM insulin was incubated alone (-○-), with 0.5 μM (-▲-), 1 μM (-▲-), or 2 μM (-▲-) G-AtBAG2, or with 2 μM G (-●-) in 50 mM HEPES buffer (pH 8.0) containing 10 mM DTT in a spectrophotometer cell at 25 °C. Data represent the mean of at least three independent experiments.
Figure 4
Figure 4
Identification of the functional domain responsible for the molecular chaperone activity of AtBAG2. (A) Schematic representation of AtBAG2 and its serial deletion variants (AtBAG2-Nt, -M, and -Ct). The circle and box indicate the UBL domain and BD, respectively. The amino acid positions of each deletion variant are indicated. G-AtBAG2 and G-AtBAG2-Nt, -M, and -Ct represent the four GST (G)-fusion constructs containing the indicated fragments of AtBAG2. (B) Results of bis-ANS-binding assays used to study the effect of heat shock on the hydrophobic domains of G-AtBAG2 and G-AtBAG2-Nt, -M, and -Ct. The samples used were 10 μM bis-ANS (-○-), 10 μM bis-ANS plus 30 μM G-AtBAG2 (-▲-), G-AtBAG2-Nt (-▲-), G-AtBAG2-M (-▲-), and G-AtBAG2-Ct (-▲-). (C) Molecular chaperone activity assay. Solutions containing either 1.67 μM MDH alone (-○-) or with 2.5 μM G-AtBAG2 (-▲-), G-AtBAG2-Nt (-▲-), G-AtBAG2-M (-▲-), and G-AtBAG2-Ct (-▲-) in 50 mM HEPES buffer (pH 7.0) were incubated in a spectrophotometer cell at 45 °C. (D) Yeast spot assay. Yeast cells transformed with pYES2-G-AtBAG2 (G-AtBAG2), pYES2-G-AtBAG2-Nt (G-AtBAG2-Nt), pYES2-G-AtBAG2-M (G-AtBAG2-M), pYES2-G-AtBAG2-Ct (G-AtBAG2-Ct), or pYES2-G (G) were grown in YPD media supplemented with 2% Gal. The transformed cells (5 × 107 cells/mL) were incubated at 55 °C for 1 h. Next, 6 μL aliquots of 10-fold serial dilutions of each cell suspension were spotted on YPD plates. The plates were incubated at 27 °C for 3 days and then photographed.
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