Cytosolic Protein Response Research Articles

SES1 positively regulates heat stress resistance in Arabidopsis

Heat stress significantly disturbs the protein folding and processing capability in plants. Molecular chaperones are vital players in unfolded/misfolded protein assembly and abiotic stress tolerance. Here, we reported SES1, which encodes an endoplasmic reticulum (ER) localized molecular chaperone, is required for Arabidopsis heat tolerance. SES1 is obviously induced by heat treatment and ses1 mutants are hypersensitive to heat stress. The unfolded protein response genes were up-regulated, while cytosolic protein response genes were down-regulated in ses1 after heat stress. Furthermore, ER stress sensor basic leucine zipper 28 (bZIP28) acts as the upstream transcriptional activator of SES1 by binding to its promoter region. These results provide new insights into heat stress responses and ER stress, and shed lights on the mechanism of SES1 in modulating heat resistance.

The roles of Arabidopsis HSFA2, HSFA4a, and HSFA7a in the heat shock response and cytosolic protein response

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.

The Arabidopsis endoplasmic reticulum associated degradation pathways are involved in the regulation of heat stress response

The Cytosolic Protein Response (CPR) in the cytosol and the Unfolded Protein Response (UPR) and ER-associated degradation (ERAD) in the endoplasmic reticulum are major pathways of the cellular proteostasis network. However, despite years of effort, how these protein quality control systems coordinated invivo remains largely unknown, particularly in plants. In this study, the roles of two evolutionarily conserved ERAD pathways (DOA10 and HRD1) in heat stress response were investigated through reverse genetic approaches in Arabidopsis. Phenotypic analysis of the mutants showed that the two ERAD pathways additively play negative roles in heat tolerance, which was demonstrated by higher survival rate and lower electrolyte leakage in the loss of function mutants compared to the wild type plants. Importantly, gene expression analysis revealed that the mutant plants showed elevated transcriptional regulation of several downstream genes, including those encoding CPR and UPR marker genes, under both basal and heat stress conditions. Finally, multiple components of ERAD genes exhibited rapid response to increasing temperature. Taken together, our data not only unravels key insights into the crosstalk between different protein quality control processes, but also provides candidate genes to genetically improve plant heat tolerance in the future.

Defects in IRE1 enhance cell death and fail to degrade mRNAs encoding secretory pathway proteins in the Arabidopsis unfolded protein response

The unfolded protein response (UPR) is a cellular response highly conserved in eukaryotes to obviate accumulation of misfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) catalyzes the cytoplasmic splicing of mRNA encoding bZIP transcription factors to activate the UPR signaling pathway. Arabidopsis IRE1 was recently shown to be involved in the cytoplasmic splicing of bZIP60 mRNA. In the present study, we demonstrated that an Arabidopsis mutant with defects in two IRE1 paralogs showed enhanced cell death upon ER stress compared with a mutant with defects in bZIP60 and wild type, suggesting an alternative function of IRE1 in the UPR. Analysis of our previous microarray data and subsequent quantitative PCR indicated degradation of mRNAs encoding secretory pathway proteins by tunicamycin, DTT, and heat in an IRE1-dependent manner. The degradation of mRNAs localized to the ER during the UPR was considered analogous to a molecular mechanism referred to as the regulated IRE1-dependent decay of mRNAs reported in metazoans. Another microarray analysis conducted in the condition repressing transcription with actinomycin D and a subsequent Gene Set Enrichment Analysis revealed the regulated IRE1-dependent decay of mRNAs-mediated degradation of a significant portion of mRNAs encoding the secretory pathway proteins. In the mutant with defects in IRE1, genes involved in the cytosolic protein response such as heat shock factor A2 were up-regulated by tunicamycin, indicating the connection between the UPR and the cytosolic protein response.

AtHsp70‐15‐deficient Arabidopsis plants are characterized by reduced growth, a constitutive cytosolic protein response and enhanced resistance to TuMV

Arabidopsis thaliana contains 18 genes encoding Hsp70s. This heat shock protein superfamily is divided into two sub-families: DnaK and Hsp110/SSE. In order to functionally characterize members of the Hsp70 superfamily, loss-of-function mutants with reduced cytosolic Hsp70 expression were studied. AtHsp70-1 and AtHsp70-2 are constitutively expressed and represent the major cytosolic Hsp70 isoforms under ambient conditions. Analysis of single and double mutants did not reveal any difference compared to wild-type controls. In yeast, SSE protein has been shown to act as a nucleotide exchange factor, essential for Hsp70 function. To test whether members of the Hsp110/SSE sub-family serve essential functions in plants, two members of the sub-family, AtHsp70-14 and AtHsp70-15, were analysed. Both genes are highly homologous and constitutively expressed. Deficiency of AtHsp70-15 but not of AtHsp70-14 led to severe growth retardation. AtHsp70-15-deficient plants were smaller than wild-type and exhibited a slightly different leaf shape. Stomatal closure under ambient conditions and in response to ABA was impaired in the AtHsp70-15 transgenic plants, but ABA-dependent inhibition of germination was not affected. Heat treatment of AtHsp70-15-deficient plants resulted in drastically increased mortality, indicating that AtHsp70-15 plays an essential role during normal growth and in the heat response of Arabidopsis plants. AtHsp70-15-deficient plants are more tolerant to infection by turnip mosaic virus. Comparative transcriptome analysis revealed that AtHsp70-15-deficient plants display a constitutive stress response similar to the cytosolic protein response. Based on these results, AtHsp70-15 is likely to be a key factor in proper folding of cytosolic proteins, and may function as nucleotide exchange factor as proposed for yeast.

The Cytosolic Protein Response as a Subcomponent of the Wider Heat Shock Response inArabidopsis

In common with a range of environmental and biological stresses, heat shock results in the accumulation of misfolded proteins and a collection of downstream consequences for cellular homeostasis and growth. Within this complex array of responses, the sensing of and responses to misfolded proteins in specific subcellular compartments involves specific chaperones, transcriptional regulators, and expression profiles. Using biological (ectopic protein expression and virus infection) and chemical triggers for misfolded protein accumulation, we have profiled the transcriptional features of the response to misfolded protein accumulation in the cytosol (i.e., the cytoplasmic protein response [CPR]) and identified the effects as a subcomponent of the wider effects induced by heat shock. The CPR in Arabidopsis thaliana is associated with the heat shock promoter element and the involvement of specific heat shock factors (HSFs), notably HSFA2, which appears to be regulated by alternative splicing and non-sense-mediated decay. Characterization of Arabidopsis HSFA2 knockout and overexpression lines showed that HSFA2 is one of the regulatory components of the CPR.