Exogenous PABA improves thermotolerance of strain <i>8213</i>.

<p>(A) Strains <i>02</i> and <i>8213</i> mycelium culture were supplied with or without 5 mg/L PABA, followed by high temperature treatment at 33°C for 48 hours, the images were taken at the indicated time post-heat stress. The control groups were cultured under regular temperature of 23°C. (B) Dose-dependent protection by PABA of mycelium intactness under heat stress at 33°C for 48 hours. Strain <i>02</i> and strain <i>8213</i> were treated with different concentrations of PABA followed by high-temperature treatment at 33°C for 48 h, and the percentage of intact mycelia was counted. The intact mycelia percentage was calculated as compared to the control group (23°C). Three independent biological replicates were performed. Data are expressed as average ± SEM. Unpaired t-tests were performed between samples with and without PABA addition within each strain, respectively, ns: P>0.05, *: P<0.05, **: P<0.01.</p

Transgenic overexpression of PABA synthase improves thermotolerance of strain <i>8213</i>.

<p>(A) Relative mRNA level of <i>Pabs</i> gene of strains <i>02</i>, <i>8213</i> and two <i>Pabs</i>-overexpressing transgenic strains <i>TB-2</i> and <i>TB-3</i> (derived from <i>8213</i>) under normal temperature (23°C) and heat stress (33°C). The mRNA of corresponding samples was extracted and analyzed after 24 hours of treatment. (B) The PABA content of strains <i>02</i>, <i>8213</i> and <i>TB-2</i> and <i>TB-3</i> under normal temperature (23°C) and heat stress (33°C) for 3 days. The PABA content of corresponding samples was extracted and measured after 3 days of treatment. (C) The mycelia growth of Strains <i>02</i>, <i>8213</i> and <i>TB-2</i> and <i>TB-3</i> under normal temperature (23°C) and heat stress (33°C). Mycelia cultures were photographed after 2 weeks of treatment. (D) The mycelia elongation of strains <i>02</i>, <i>8213</i> and <i>TB-2</i> and <i>TB-3</i> under normal temperature (23°C) and heat stress (33°C). The mycelia length is measured after 14 and 21 days of treatment. Three independent biological replicates were performed for each analysis. Data are expressed as average ± SEM. Unpaired t-tests were performed between strain <i>8213</i> and all other strains as indicated within each treatment condition, ns: P>0.05, *: P<0.05, **: P<0.01.</p

Identification and classification of differentially expressed proteins between <i>02</i> and <i>8213</i>.

<p>(A) Venn diagram showing 20 up-regulated and 5 down-regulated proteins among 48 identified proteins comparing <i>02</i>-HS with <i>02</i>-NS. Functional classification of those 25 heat stress induced proteins is shown on the bottom. (B) Heat-map is shown to indicate relative protein abundance among the 4 samples. The abundance of each protein spot in <i>02</i>-NS was given a reference value of 1.0, the abundance of that spot from <i>02</i>-HS, <i>8213</i>-NS and <i>8213</i>-HS were transformed into relative value. The different colors correspond to the values of protein level changes as indicated by the bar at the bottom of the heat map. Venn diagrams present the two-step filtering process leading to 4 candidates to further select out the more important candidate proteins for functional studies (see text). The four asterisks mark the proteins that are upregulated by heat-stress in thermotolerant strain <i>02</i>, not upregulated by heat stress in thermo-sensitive strain <i>8213,</i> and expressed at lower levels in thermo-sensitive strain <i>8213</i> than in the thermotolerant strain <i>02</i> with or without heat-stress. <i>02</i>-NS: <i>02</i> non-stressed (23°C/24 h); <i>02</i>-HS: <i>02</i> heat-stressed (33°C/24 h); <i>8213</i>-NS: <i>8213</i> non-stressed (23°C/24 h); <i>8213</i>-HS: strain <i>8213</i> heat-stressed (33°C/24 h).</p

PABA reduces H₂O₂ accumulation in heat stressed strains <i>02</i> and <i>8213</i>.

<p>(A) Time course of accumulation of H₂O₂ in the mycelia of strains <i>02</i>, <i>8213</i>, <i>TB-2</i> and <i>TB-3</i> under heat stress (33°C). Strains <i>02</i>, <i>TB-2</i> and <i>TB-3</i>, which produce more PABA content than strain <i>8213</i>, have less H₂O₂ accumulation under parallel conditions. (B) Effects of PABA and PABA synthase inhibitor sulfanilamide on H₂O₂ accumulation. Strain <i>02</i> and strain <i>8213</i> were cultured on PDA medium with 1 mM PABA or 0.1 mM sulfanilamide for 6 days, then were subjected to high temperature treatment (33°C) for 48 hours as indicated, followed by H₂O₂ content measurement. Three independent biological replicates were performed for each analysis. Data are expressed as average ± SEM. Unpaired t-tests were performed between control sample and sulfanilamide treated sample or PABA treated sample within each strain, ns: P>0.05, *: P<0.05, **: P<0.01.</p

Exogenous PABA improves thermotolerance of strain <i>8213</i>.

<p>(A) Strains <i>02</i> and <i>8213</i> mycelium culture were supplied with or without 5 mg/L PABA, followed by high temperature treatment at 33°C for 48 hours, the images were taken at the indicated time post-heat stress. The control groups were cultured under regular temperature of 23°C. (B) Dose-dependent protection by PABA of mycelium intactness under heat stress at 33°C for 48 hours. Strain <i>02</i> and strain <i>8213</i> were treated with different concentrations of PABA followed by high-temperature treatment at 33°C for 48 h, and the percentage of intact mycelia was counted. The intact mycelia percentage was calculated as compared to the control group (23°C). Three independent biological replicates were performed. Data are expressed as average ± SEM. Unpaired t-tests were performed between samples with and without PABA addition within each strain, respectively, ns: P>0.05, *: P<0.05, **: P<0.01.</p

2-D electrophoresis of protein extracts from <i>02</i> and <i>8213</i> with or without heat stress (33°C/24 h).

<p>(A) Representative 2-DE gels of mushrooms, identifying 73 proteins with a greater than 2-fold difference after high-temperature treatment (p<0.05). Molecular weight (MW) in kilodaltons and pI of proteins are indicated on the left and top of the gel, respectively. (B) Close-up view of some differentially expressed proteins spots. Three independent replicates were performed. <i>02</i>-NS: <i>02</i> non-stressed (23°C/24 h); <i>02</i>-HS: <i>02</i> heat-stressed (33°C/24 h); <i>8213</i>-NS: <i>8213</i> non-stressed (23°C/24 h); <i>8213</i>-HS: strain <i>8213</i> heat-stressed (33°C/24 h).</p

PABA mediates the accumulation of defense-related proteins in <i>02</i>, <i>8213</i> and <i>TB-2</i> under heat stress.

<p>(A) Defense-related proteins HSPs and Chitinase accumulated more in thermotolerant strain <i>02</i> than in thermo-sensitive strain <i>8213</i> under heat stress (33°C). (B) Exogenous PABA (1 mM) application increases accumulation of HSPs and Chitinase in <i>8213</i> under heat stress (33°C). (C) PABA synthase inhibitor Sulfanilamide (0.1 mM) decreases accumulation of HSPs and Chitinase in <i>02</i> under heat stress (33°C). D) <i>Pabs</i>-overexpressing transgenic line <i>TB-2</i> accumulates more HSPs and Chitinase than the parent strain <i>8213</i> under heat stress (33°C). Three independent biological replicates were performed for each sample.</p

Schematic model for the role of PABA in enhancing thermotolerance of mushroom.

<p>See Discussion for details.</p

PABA increases, while sulfanilamide decreases, the activity of Catalase and SOD in <i>02</i>, <i>8213</i> and <i>TB-2</i>.

<p>Mushroom strains, as indicated, were treated with 1(33°C) for 2 days. The enzymes activities of Catalase and SOD were measured immediately after heat stress. Three independent biological replicates were performed for each analysis. Data are expressed as average ± SEM. Unpaired t-tests were performed as indicated in the figure, ns: P>0.05, *: P<0.05, **: P<0.01, Sul: sulfanilamide.</p

Thermotolerant strain <i>02</i> responded to heat stress with more PABA synthesis than strain <i>8213</i>.

<p>(A) MS/MS analysis identifies the protein spot 36 to be mushroom PABA synthase. (B) Differential transcription of PABA synthase gene <i>Pabs</i> in strain <i>02</i> and strain <i>8213</i> with or without heat stress. Strain <i>02</i> and strain <i>8213</i> were treated with high temperature at 33°C for the indicated time and the mRNA level of <i>Pabs</i> was quantified by real-time PCR. Three independent biological replicates were performed. Data are expressed as average ± SEM. Unpaired t-tests were performed between high temperature treatment samples and control samples (without treatment) within each strain, respectively, ns: P>0.05, *: P<0.05, **: P<0.01. (C) Differential protein accumulation of PABA synthase in strain <i>02</i> and strain <i>8213</i> after heat stress. Strain <i>02</i> and strain <i>8213</i> were treated with high temperature at 33°C for the indicated time, and PABA synthase protein levels were measured by western blot as indicated. (D) Differential accumulation of PABA content in strain <i>02</i> and strain <i>8213</i> after heat stress. Strain <i>02</i> and strain <i>8213</i> were treated with high temperature at 33°C for the indicated time and the content of PABA was measured by HPLC. Three independent biological replicates were performed. Data are expressed as average ± SEM.</p