Effect of HDAC inhibitor TSA on haploid embryogenesis [dataset 1]
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ABSTRACT: Haploid embryos can be induced from cultured immature pollen following a stress treatment. In Brassica napus, application of the histone/lysine deacetylase (HDAC/KDAC) inhibitor trichostatin A (TSA) to pollen cultures enhances the production of differentiated embryos and embryogenic callus when applied together with heat stress (Li et al., 2014). To identify genes associated with the induction of B. napus haploid embryogenesis, we compared the transcriptomes of untreated pollen cultures and pollen cultures treated with either heat-stress or heat-stress plus TSA.
Project description:Haploid embryos can be induced from cultured immature pollen following stress treatment. In Brassica napus, the application of the histone/lysine deacetylase (HDAC/KDAC) inhibitor trichostatin A (TSA) to pollen cultures enhances the production of differentiated embryos and embryogenic callus when applied together with heat stress (Li et al., 2014). To identify genes associated with the induction of haploid embryogenesis and to investigate which genes may be responsible for TSA-induced lipid and starch accumulation in the embryogenic structures, we compared the transcriptomes of pollen cultures treated with heat stress and 0.05 µM TSA to those of untreated pollen cultures, both at two days.
Project description:The haploid multicellular male gametophyte of plants, the pollen grain, is a terminally differentiated structure whose function ends at fertilization. Unlike pollen grains, the immature gametophyte retains its capacity for totipotent growth when cultured in vitro. Haploid embryo production from cultured immature male gametophytes is a widely used plant breeding and propagation technique that was described nearly 50 years ago, but one that is poorly understood at the mechanistic level. Using a chemical approach, we show that the switch to haploid embryogenesis is controlled by the activity of histone deacetylases (HDACs). Blocking HDAC activity with trichostatin A (TSA) in cultured immature male gametophytes of Brassica napus leads to a large increase in the proportion of cells that switch from pollen to embryogenic growth. Embryogenic growth is enhanced by, but not dependent on, the high temperature stress that is normally used to induce haploid embryogenesis in B. napus. The immature male gametophyte of Arabidopsis thaliana, which is recalcitrant for haploid embryo development in culture, also forms embryogenic cell clusters after TSA treatment. TSA treatment of immature male gametophytes for as little as eight hours was accompanied by hyperacetylation of histones H3 and H4, and by the upregulation of genes involved in cell-cycle progression, the auxin pathway and cell wall catabolism pathways. We propose that the totipotency of the immature male gametophyte in planta is kept in check by an HDAC-dependent mechanism, and that high temperature or other stresses used to induce haploid embryo development in culture impinge on this HDAC-dependent pathway. 8 samples were analyzed. We generated the following pairwise comparisons between treatment and the corresponding mock treatment: TSA+CHX (2 replicates) vs CHX (2 replicates); TSA (2 replicates) vs DMSO (2 replicates).
Project description:The haploid multicellular male gametophyte of plants, the pollen grain, is a terminally differentiated structure whose function ends at fertilization. Unlike pollen grains, the immature gametophyte retains its capacity for totipotent growth when cultured in vitro. Haploid embryo production from cultured immature male gametophytes is a widely used plant breeding and propagation technique that was described nearly 50 years ago, but one that is poorly understood at the mechanistic level. Using a chemical approach, we show that the switch to haploid embryogenesis is controlled by the activity of histone deacetylases (HDACs). Blocking HDAC activity with trichostatin A (TSA) in cultured immature male gametophytes of Brassica napus leads to a large increase in the proportion of cells that switch from pollen to embryogenic growth. Embryogenic growth is enhanced by, but not dependent on, the high temperature stress that is normally used to induce haploid embryogenesis in B. napus. The immature male gametophyte of Arabidopsis thaliana, which is recalcitrant for haploid embryo development in culture, also forms embryogenic cell clusters after TSA treatment. TSA treatment of immature male gametophytes for as little as eight hours was accompanied by hyperacetylation of histones H3 and H4, and by the upregulation of genes involved in cell-cycle progression, the auxin pathway and cell wall catabolism pathways. We propose that the totipotency of the immature male gametophyte in planta is kept in check by an HDAC-dependent mechanism, and that high temperature or other stresses used to induce haploid embryo development in culture impinge on this HDAC-dependent pathway.
Project description:Somatic embryogenesis (SE) exemplifies the unique developmental plasticity of plant cells. The regulatory processes, including epigenetic modifications controlling embryogenic reprogramming of cell transcriptome, have just started to be revealed. To identify the genes of histone acetylation-regulated expression in SE, we analyzed global transcriptomes of Arabidopsis explants undergoing embryogenic induction in response to treatment with histone deacetylase inhibitor, trichostatin A (TSA). The TSA-induced and auxin (2,4-dichlorophenoxyacetic acid; 2,4-D)-induced transcriptomes were compared. RNA-seq results revealed the similarities of the TSA- and auxin-induced transcriptomic responses that involve extensive deregulation, mostly repression, of the majority of genes. Within the differentially expressed genes (DEGs), we identified the master regulators (transcription factors TF) of SE, genes involved in biosynthesis, signaling, and polar transport of auxin and NITRILASE-encoding genes of the function in indole-3-acetic acid (IAA) biosynthesis. TSA-upregulated TF genes of essential functions in auxin-induced SE, included LEC1/LEC2, FUS3, AGL15, MYB118, PHB, PHV, PLTs, and WUS/WOXs. The TSA-induced transcriptome revealed also extensive upregulation of stress-related genes, including those related to stress hormone biosynthesis. In line with transcriptomic data, TSA-induced explants accumulated salicylic acid (SA) and abscisic acid (ABA), suggesting the role of histone acetylation (Hac) in regulating stress hormone-related responses during SE induction. Since mostly the adaxial side of cotyledon explant contributes to SE induction, we also identified organ polarity-related genes responding to TSA treatment, including AIL7/PLT7, RGE1, LBD18, 40, HB32, CBF1, and ULT2. Analysis of the relevant mutants supported the role of polarity-related genes in SE induction. The study results provide a step forward in deciphering the epigenetic network controlling embryogenic transition in somatic cells of plants.
Project description:High temperature stress results in yield loss and alterations to seed composition during seed filling in oilseed rape (Brassica napus). However, the mechanism underlying this heat response is poorly understood. In this study, we employed a microarray analysis with silique walls and seeds from the developing siliques (20 days after flowering) of Brassica napus that had undergone heat stress. Two-condition experiment, control vs heat stress, 2 time points
Project description:High temperature stress results in yield loss and alterations to seed composition during seed filling in oilseed rape (Brassica napus). However, the mechanism underlying this heat response is poorly understood. In this study, we employed a microarray analysis with silique walls and seeds from the developing siliques (20 days after flowering) of Brassica napus that had undergone heat stress.
Project description:Pollen development is one of the most heat-sensitive developmental stages in a wide range of crops. Our longer-term goal is to understand the mechanism how starch metabolism in maturing pollen grains of the Solanaceae family contributes to maintaining higher pollen quality under heat-stress conditions. The specific aim of the suggested proposal is to characterize N. sylvestris WT and mutant (starch-deficient) transcriptomes during microgametogenesis under ambient and heat-stress conditions. Expression profiles of maturing microspores derived from flower buds at developmental stage of 4 to 2 days before flower opening will be obtained. Pollen was derived from WT and mutant plants exposed to either ambient or heat-stress conditions (exposing the plants to 45oC for 2.5 hours). Keywords: Loop design
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:Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate and energy metabolism. Ethephon-pretreatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.