Abstract
Previously, we found that Arabidopsis plants transformed with a construct containing the promoter of Oshsp17.3 from rice fused to the β-glucuronidase gene (GUS), Oshsp17.3Pro::GUS (Oshsp17.3p), showed a GUS signal after heat shock (HS) or azetidine-2-carboxylic acid (AZC) treatment. HS and AZC trigger the heat shock response (HSR) and cytosolic protein response (CPR), respectively, in the cytosol by modulating specific heat shock factor (HSF) activity. Here we further identified that AtHSFA2 (At2g26150), AtHSFA7a (At3g51910), AtHSFB2a (At5g62020), and AtHSFB2b (At4g11660) are HS- and AZC-inducible; AtHSFA4a (At4g18880) is AZC-inducible; and AtHSFA5 (At4g13980) is less AZC- and HS-inducible. To investigate the roles of these 6 AtHSFs in the HSR or CPR, we crossed two independent Oshsp17.3p transgenic Arabidopsis plants with the AtHSF-knockout mutants athsfa2 (SALK_008978), athsfa4a (GABI_181H12), athsfa5 (SALK_004385), athsfa7a (SALK_080138), athsfb2a (SALK_137766), and athsfb2b (SALK_047291), respectively. As compared with the wild type, loss-of-function mutation of AtHSFA2, AtHSFA4a, and AtHSFA7a decreased HS and AZC responsiveness, so these 3 AtHSFs are essential for the HSR and CPR. In addition, loss-of-function results indicated that AthsfB2b is involved in regulating the HSR in Arabidopsis. Furthermore, analysis of the relative GUS activity of two double knockout mutants, athsfA2/athsfA4a and athsfA2/athsfA7a, revealed that AtHSFA2, AtHSFA4a, and AtHSFA7a function differentially in the HSR and CPR. Transcription profiling in athsf mutants revealed positive or negative transcriptional regulation among the 6 AtHSFs in Arabidopsis plants under HS and AZC conditions. Tunicamycin treatment demonstrated that these 6 AtHSFs are not involved in the unfolded protein response.
Highlights
Protein homeostasis is crucial for maintaining normal cellular function
AtHSFA2, AtHSFA4a, and AtHSFA7a genes function differentially in response to heat and azetidine-2-carboxylic acid (AZC) stress To investigate whether the AtHSFs examined are involved in the heat shock (HS) or AZC responsiveness, Two independent Oshsp17.3Pro::GUS transgenic plants, Oshsp17.3p5 and Oshsp17.3p11, were separately crossed with athsfA2, athsfA4a, athsfA5, athsfA7a, athsfB2a, and athsfB2b mutants
Lines Oshsp17.3p5/athsfA2, Oshsp17.3p5/ athsfA4a, Oshsp17.3p5/athsfA5, Oshsp17.3p5/athsfA7a, Oshsp17.3p5/athsfB2a, Oshsp17.3p5/athsfB2b, Oshsp17.3p5/athsfA2/athsfA4a, Oshsp17.3p5/athsfA2/ athsfA7a, Oshsp17.3p11/athsfA2, Oshsp17.3p11/athsfA4a, Oshsp17.3p11/athsfA5, Oshsp17.3p11/athsfA7a, Oshsp17.3p11/athsfB2a, Oshsp17.3p11/athsfB2b, Oshsp17.3p11/athsfA2/athsfA4a, and Oshsp17.3p11/ athsfA2/athsfA7a were obtained for analyzing HS and AZC responsiveness
Summary
Protein homeostasis is crucial for maintaining normal cellular function. Extremes in environmental factors can result in stressful conditions that inevitably damage proteins directly or cause cells to synthesize misfolded proteins, which can lead to perturbed cell function and stress-induced cell death. Plants have evolved an extensive network of chaperone systems to restore protein folding or to remove irreversibly. Accumulation of unfolded proteins within cells, eliciting compartment-specific chaperones and pathways, is termed the unfolded protein response (UPR). The UPR initiates the dissociation of the endoplasmic reticulum (ER) chaperone, immunoglobulin binding protein, and ER master sensors, such as inositol-requiring 1 and protein kinase R-like ER kinase, to activate downstream effectors to restore protein homeostasis in the lumen of the ER. A cytosolic process, the cytoplasmic protein response (CPR), increases the synthesis of molecular chaperones such as heat shock proteins (HSPs).
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