Cytosol-Localized Heat Shock Factor-Binding Protein, AtHSBP, Functions as a Negative Regulator of Heat Shock Response by Translocation to the Nucleus and Is Required for Seed Development in Arabidopsis      

Abstract

Heat shock response (HSR) is a universal mechanism in all organisms. It is under tight regulation by heat shock factors (HSFs) and heat shock proteins (HSPs) after heat shock (HS) to prevent stress damage. On the attenuation of HSR, HSP70 and HSF Binding Protein1 (HSBP1) interact with HSF1 and thus dissociate trimeric HSF1 into an inert monomeric form in humans. However, little is known about the effect of HSBP with thermal stress in plants. This report describes our investigation of the role of AtHSBP in Arabidopsis (Arabidopsis thaliana) by genetic and molecular approaches. AtHSBP was heat inducible and ubiquitously expressed in all tissues; AtHSBP was also crucial for seed development, as demonstrated by AtHSBP-knockout lines showing seed abortion. Thermotolerance results showed that AtHSBP participates in acquired thermotolerance but not basal thermotolerance and is a negative regulator of HSR. Subcellular localization revealed that the cytosol-localized AtHSBP translocated to the nucleus in response to HS. Protoplast two-hybrid assay results confirmed that AtHSBP interacts with itself and with the HSFs, AtHSFA1a, AtHSFA1b, and AtHSFA2. AtHSBP also negatively affected AtHSFA1b DNA-binding capacity in vitro. Quantitative polymerase chain reaction and western-blot analysis demonstrated that altered levels of AtHSBP lead to differential HSP expression, mainly during the recovery from HS. These studies provide a new insight into HSBP in plants and reveal that AtHSBP is a negative regulator of HSR and required for seed development.

The heat-stress factor HSFA6b connects ABA signaling and ABA-mediated heat responses

“…To generate the constructs for BiFC analysis, the RT-PCR-amplified HSFs were cloned into a pCR8/GW/TOPO vector and then recombined into pEarleyGate201-YN or pEarleyGate202-YC vectors (Lu et al, 2010). An amount of 2 3 10 4 protoplasts was transfected with 10 to 20 mg DNA, then incubated at 22°C for 16 to 24 h. The reconstituted YFP signals were observed by confocal microscopy (TCS SP5; Leica) as described previously (Hsu et al, 2010).…”

“…A CaMV 35S promoter was cloned into the P GAL4-UASx4 ::GUS vector, which resulted in P 35S ::GUS construct, to be used as a positive control. The transactivation assay involved cotransfection with a mixture of 15 mg effector (P 35S ::AREB1-3XFLAG, P 35S ::HSFA3-3XFLAG, P 35S ::HSFA6a-3XFLAG, or P 35S ::HSFA6b-3XFLAG) and reporter plasmids, using 5 mg MTC-301 luciferase plasmid as an internal control to normalize the transfection efficiencies (Ehlert et al, 2006;Hsu et al, 2010). The protoplasts were incubated for 12 to 16 h, then treated without or with 10 mM ABA for 12 h. Luciferase activity was analyzed by use of Luciferase Assay buffer according to the technical manual (Promega), and GUS activity assay was performed as described (Yoo et al, 2007).…”

“…When stress is relieved, the HSR is attenuated by excess HSP70 and other proteins that repress the transcriptional activity of HSFs by binding to them and converting HSFs back to the original inactive form. AtHSBP, an HSF-binding protein in Arabidopsis (Arabidopsis thaliana), can reduce the DNA binding capacity of HSFs and function as a negative regulator of the HSR (Hsu et al, 2010).…”

The Protein Phosphatase RCF2 and Its Interacting Partner NAC019 Are Critical for Heat Stress–Responsive Gene Regulation and Thermotolerance in Arabidopsis

“…AtCAM3 plays a role in the Ca 2+ -calmodulin heat stress signal transduction pathway in Arabidopsis (Zhang et al, 2009). Furthermore, AtHSBP interacts with HSFA1a, HSFA1b, and HSFA2 and negatively regulates the binding of HSFA1b to a heat stress element in vitro, thereby functioning as a negative regulator for heat stress responses in Arabidopsis (Hsu et al, 2010). A KH domain-containing putative RNA binding protein, RCF3 (for regulator of CBF gene expression 3), acts as a negative regulator of heat stress-responsive gene expression and thermotolerance in Arabidopsis (Guan et al, 2013a).…”

Expression of Heat Shock Proteins by Heat Stress in Soybean

“…Chaperones and HSPs have functions which restoration of mis-folding proteins and minimizing damages for maintenance plant homeostasis (Boston et al 1996;Bukau et al 2006;Pratt et al 2010). Also, these stress response systems are regulated by HSFs which has DNA binding sites (Schöffl et al 1998;Nover et al 2001;Hsu et al 2010).…”