Project description:Transcriptional regulation is a key aspect of environmental stress responses. Heat stress (HS) induces transcriptional memory that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crops is an important breeding goal. However, not all HS-inducible genes show sustained induction/transcriptional memory and it is unclear what distinguishes memory and non-memory genes. To address this issue and understand the (epi-) genome architecture in transcriptional memory after HS, we investigated genome-wide target genes of the two key memory heat shock transcription factors, HSFA2 and HSFA3. We determined the binding kinetics of these factors to their target genes and asked whether genes that show sustained induction of transcription carry specific features that allow prediction and potentially engineering of memory gene behaviour. HSFA2 and HSFA3 show near identical binding patterns. In vitro binding strength as determined by DAP-seq analysis correlates strongly with in vivo binding strength, confirming the importance of sequence features. However, no single distinctive sequence motif appears to be required for memory behaviour. Instead, HS memory genes are characterized by a combination of features: low expression levels in the absence of HS, chromatin environment and an enrichment of H3K4 methylation after HS. Our findings are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. In summary, our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants. They will contribute to the prediction and engineering of genes with transcriptional memory.
Project description:Transcriptional regulation is a key aspect of environmental stress responses. Heat stress (HS) induces transcriptional memory that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crops is an important breeding goal. However, not all HS-inducible genes show sustained induction/transcriptional memory and it is unclear what distinguishes memory and non-memory genes. To address this issue and understand the (epi-) genome architecture in transcriptional memory after HS, we investigated genome-wide target genes of the two key memory heat shock transcription factors, HSFA2 and HSFA3. We determined the binding kinetics of these factors to their target genes and asked whether genes that show sustained induction of transcription carry specific features that allow prediction and potentially engineering of memory gene behaviour. HSFA2 and HSFA3 show near identical binding patterns. In vitro binding strength as determined by DAP-seq analysis correlates strongly with in vivo binding strength, confirming the importance of sequence features. However, no single distinctive sequence motif appears to be required for memory behaviour. Instead, HS memory genes are characterized by a combination of features: low expression levels in the absence of HS, chromatin environment and an enrichment of H3K4 methylation after HS. Our findings are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. In summary, our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants. They will contribute to the prediction and engineering of genes with transcriptional memory.
Project description:HSFA1s are a gene family of HSFA1 with four members, HSFA1a, HSFA1b, HSFA1d, and HSFA1e. HSFA1s are the master regulators of heat shock response. As a part of the heat shock response, HSFA2 can prolong the heat shock response and amplify the heat shock response in response to repeat heat shock. To identify the heat-shock-responsive genes differentially regulated by HSFA1s and HSFA2, we compared the transcriptomic differences of plants containing only constitutively expressed HSFA1s or HSFA2 after heat stress. hsfa2 (the KO mutant of HSFA2, Col-0 background) and A2QK-10 (CaMV 35S:HSFA2 in QK mutant; QK is HSFA1a/b/d/e quadruple KO mutant) were used to compare the difference of heat shock response when plants lack HSFA1s or HSFA2. The aim is to find the HSFA1s- and HSFA2-preferred regulating genes after heat stress. As the control samples, wild type is the plant with normal heat shock response, and QK (HSFA1s KO mutant, Col-0 and Ws mixed background) is the plant that lost the heat shock response controlled by HSFA1s.
Project description:HSFA1s are a gene family of HSFA1 with four members, HSFA1a, HSFA1b, HSFA1d, and HSFA1e. HSFA1s are the master regulators of heat shock response. As a part of the heat shock response, HSFA2 can prolong the heat shock response and amplify the heat shock response in response to repeat heat shock. To identify the heat-shock-responsive genes differentially regulated by HSFA1s and HSFA2, we compared the transcriptomic differences of plants containing only constitutively expressed HSFA1s or HSFA2 after heat stress.
Project description:The expression of heat-shock proteins (Hsps) induced by a non-lethal heat treatment confers acquired thermotolerance (AT) to organisms against a subsequent challenge of otherwise lethal temperature. After stress signal lifted, AT gradually decayed with the decline of Hsps during recovery period. The duration of AT may be critical for sessile organisms, such as plants, to survive repeated heat stress in the environment. To identify heat-induced genes involved in duration of AT, we took a reverse-genetics approach by screening for Arabidopsis T-DNA insertion mutants that show decreased thermotolerance after a long recovery at non-stress condition following a conditioning treatment. Among the tested mutants corresponding to 47 genes, only the HsfA2 knockout mutant showed significant phenotype. The mutant plants were more sensitive to severe heat stress than the wild type after long but not short recovery following a pretreatment at 37oC, which can be complemented by introducing a wild-type copy of the gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in the absence of HsfA2. Significant decline of the transcript levels of several highly heat-induced genes was observed in the HsfA2 knockout plants after a 4-h recovery or after 2 h of prolonged heat stress. Immunoblot anlysis showed that Hsa32 and class I small Hsp were lower in the mutant than in the wild type after a long recovery. Our results suggest that HsfA2 as a heat-induced transactivator sustains the post-stress expression of Hsp genes and extends the duration of AT in Arabidopsis. Keywords: heat shock response
Project description:Male reproductive tissues are more sensitive to heat stress compared to vegetative tissues, however the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection and recovery from heat stress. HsfA2 has been characterized as co-activator of HsfA1a in tomato and is considered as one of the major Hsfs accumulating in response to elevated temperatures. The role of HsfA2 in heat stress response of different tissues was examined by exploring the composition and structure of the tissue-specific regulatory networks in transgenic tomato plants with suppressed HsfA2 expression (A2AS). Transcriptome analysis revealed that HsfA2 acts in condition- and tissue-specific manner and that only a subset of heat stress induced genes require HsfA2 for higher expression. Remarkably, although HsfA2 is not essential for thermotolerance in seedlings and flowering plants, it is required for maintenance pollen viability under stress conditions. We show that the activation of Hsf networks is important for the developmentally regulated priming of heat stress response occurring at early stages of anther and pollen development. Thereby, HsfA2 is involved in pollen thermotolerance by directly regulating heat stress responsive genes but also by stimulating the synthesis of molecular chaperones under non-stress conditions.
Project description:The expression of heat-shock proteins (Hsps) induced by a non-lethal heat treatment confers acquired thermotolerance (AT) to organisms against a subsequent challenge of otherwise lethal temperature. After stress signal lifted, AT gradually decayed with the decline of Hsps during recovery period. The duration of AT may be critical for sessile organisms, such as plants, to survive repeated heat stress in the environment. To identify heat-induced genes involved in duration of AT, we took a reverse-genetics approach by screening for Arabidopsis T-DNA insertion mutants that show decreased thermotolerance after a long recovery at non-stress condition following a conditioning treatment. Among the tested mutants corresponding to 47 genes, only the HsfA2 knockout mutant showed significant phenotype. The mutant plants were more sensitive to severe heat stress than the wild type after long but not short recovery following a pretreatment at 37oC, which can be complemented by introducing a wild-type copy of the gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in the absence of HsfA2. Significant decline of the transcript levels of several highly heat-induced genes was observed in the HsfA2 knockout plants after a 4-h recovery or after 2 h of prolonged heat stress. Immunoblot anlysis showed that Hsa32 and class I small Hsp were lower in the mutant than in the wild type after a long recovery. Our results suggest that HsfA2 as a heat-induced transactivator sustains the post-stress expression of Hsp genes and extends the duration of AT in Arabidopsis. Experiment Overall Design: Total RNA was isolated from the seedlings of 5-d old wild-type and HsfA2 knockout mutant seedlings (a pool of about 100 plants per treatment in duplicates) harvested immediately after heat shock treatment. In this experiment, total 12 chips were used, 1 each for 2 biological replicates of the control and HS-treated samples for the wild type and mutant plants.
Project description:Male reproductive tissues are more sensitive to heat stress compared to vegetative tissues, however the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection and recovery from heat stress. HsfA2 has been characterized as co-activator of HsfA1a in tomato and is considered as one of the major Hsfs accumulating in response to elevated temperatures. The role of HsfA2 in heat stress response of different tissues was examined by exploring the composition and structure of the tissue-specific regulatory networks in transgenic tomato plants with suppressed HsfA2 expression (A2AS). Transcriptome analysis revealed that HsfA2 acts in condition- and tissue-specific manner and that only a subset of heat stress induced genes require HsfA2 for higher expression. Remarkably, although HsfA2 is not essential for thermotolerance in seedlings and flowering plants, it is required for maintenance pollen viability under stress conditions. We show that the activation of Hsf networks is important for the developmentally regulated priming of heat stress response occurring at early stages of anther and pollen development. Thereby, HsfA2 is involved in pollen thermotolerance by directly regulating heat stress responsive genes but also by stimulating the synthesis of molecular chaperones under non-stress conditions. 8 samples