Project description:Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2 (1-3). Here, we report the identification of ETHYLENE INSENSITIVE5 as the 5’?3’ exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response. Keywords: total RNA profiling, proteolysis, plant hormoine, ethylene
Project description:thylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2 (1-3). Here, we report the identification of ETHYLENE INSENSITIVE5 as the 5'->3' exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response. Keywords: Arabidopsis growth regulation signal transduction
Project description:Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2 (1-3). Here, we report the identification of ETHYLENE INSENSITIVE5 as the 5â?3â exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response. Experiment Overall Design: Cold-treated seeds in MS plates were placed in chambers at 24oC in the dark with hydrocarbon-free airflow for three days, after which some of the chambers were connected to ethylene gas at 10 ppm while the others remained on air treatment. Four hours later the seedlings of each plate were quickly collected and frozen in liquid nitrogen. Total RNA was prepared from both air-treated and ethylene-treated etiolated seedlings of Arabidopsis Col-0, ein2-5 and ein5-1 using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Biotinylated target RNA was prepared from 16 ?g of total RNA using the procedure described by the manufacturer (Affymetrix, Santa Clara, CA). Briefly, a primer encoding a T7 RNA polymerase promoter fused to (dT)24 (Genset Oligos, La Jolla, CA) was used to prime double-stranded cDNA synthesis using the SuperScript Choice System (Life Technologies). The resulting cDNA was transcribed in vitro using the BioArray High-Yield RNA Tran-script Labeling Kit (Enzo Biochem, New York, NY) in the presence of biotinylated UTP and CTP to produce biotinylated target complementary RNA (cRNA). The labeled target cRNA was purified, fragmented, and hybridized to Arabidopsis microoarrays (Affymetrix whole genome tiling, and ATH1 gene expression arrays) according to protocols provided by the manufacturer in a hybridization oven model 640 (Affymetrix, Santa Clara, CA). The arrays were washed and stained with streptavidin-phycoerythrin using a GeneChip Fluidics Station model 400 and then scanned with a Gene Array Scanner (Hewlett-Packard, Palo Alto, CA). Scanned images were processed and quantified using GeneChip Suite 3.2. Genespring software (Silicon Genetics, Redwood City, CA) was used to manage and filter the array data. Each measurement was divided by the 50.0th percentile of all measurements in that sample. The percentile was calculated with all normalized measurements above 10. For samples where the bottom tenth percentile was less than the negative of the 50.0th percentile, it was used as a background, and subtracted from all the other genes first. Experiments were carried out in duplicate and hybridized to two sets of expression chips. Expression values were analyzed using GeneSpring⢠software version 4.2 (Silicon Genetics, Redwood, California).
Project description:Ethylene is a gaseous plant hormone that regulates plant growth and development. Broad reprogramming of gene expression is required for ethylene responses. The primary ethylene transcription factor (TF) ETHYLENE INSENSITIVE3 (EIN3) drives expression of secondary TFs including the ETHYLENE RESPONSE DNA-BINDING FACTORS (EDFs), but the role of the EDFs within the ethylene genome regulatory network is not understood. Here, we describe an investigation into the function of the EDFs in ethylene signalling and hormonal cross-regulation. We determined the target genes and binding dynamics of EDFs 1, 2, 3 during an ethylene response and the effects of edf1234 quadruple mutation on gene expression. The EDFs and EIN3 shared a large proportion of their target genes but had different functions. The EDFs were associated with repression of target genes, but this was superseded by activation when EIN3 bound the same genes. Genes important in other hormone signalling pathways, in particular abscisic acid (ABA), were targets of the EDFs. This demonstrates how ethylene engages hormonal cross-regulation to repress genes in competing signalling pathways and prioritize itself.
Project description:The plant hormone ethylene is involved in plant developmental, stress and environmental signalling. The ethylene signalling pathway is modulated by the master transcription factor EIN3. Here we examined the involvement of proteasome-associated UPL3 and UPL4 ubiquitin ligases in ethylene-responsive gene expression. We reveal that in absence of functional UPL3 and UPL4, plants show constitutive and enhanced activation or repression of ethylene-responsive genes in an EIN3-dependent manner. Ten-day old Arabidopsis thaliana seedlings of wild-type Col-0, mutant upl3 upl4, mutant ein3-1, and mutant upl3 upl4 ein3-1 were grown on MS media in an environmental chamber with 16/8 hour day/night light regime (120 mol m-2 s-1 light intensity) and 22 degrees Celsius. Seedlings were then transferred to 6-well plates and floated on the surface of water or 50 uM 1-aminocyclopropane-1-carboxylic acid (ACC). After 3 hours seedlings were harvested and for each treatment ~50 seedlings were pooled together into a single biological repeat. In total three independent biological repeats were collected. After harvesting seedlings were briefly dried on tissue and immediately frozen in liquid nitrogen until further analysis.
Project description:Jasmonate and ethylene are two important plant hormones contributing plant resistance to biotic stresses synergistically. Our experimental results showed that two ethylene activated transcription factors (EIN3/EIL1) integrated both jasmonate and ethylene signaling in multiple developmental and defense events. To understand the importance of EIN3/EIL1 in jasmonate signaling, we utilized microarray analysis to identify how much the jasmonate regulated genes are governed by EIN3/EIL1. Our results indicated that EIN3/EIL1 act sequentially in a cascade of transcriptional regulation initiated by jasmonate.
Project description:Jasmonate and ethylene are two important plant hormones contributing plant resistance to biotic stresses synergistically. Our experimental results showed that two ethylene activated transcription factors (EIN3/EIL1) integrated both jasmonate and ethylene signaling in multiple developmental and defense events. To understand the importance of EIN3/EIL1 in jasmonate signaling, we utilized microarray analysis to identify how much the jasmonate regulated genes are governed by EIN3/EIL1. Our results indicated that EIN3/EIL1 act sequentially in a cascade of transcriptional regulation initiated by jasmonate. Compare the JA regulated genes in Col-0, coi1-2 and ein3 eil1 to identify how much transcripts are dependent on COI1 or EIN3/EIL1
Project description:Ethylene is a gaseous hormone that plays important roles in plant growth, development and stress responses. EIN3 was identified as a plant-specific transcription factor and its protein level rapidly increases upon ethylene treatment. We found that activation of EIN3 and EIN3-like 1 (EIL1) is both necessary and sufficient for ethylene-induced enhancement of seedling greening, as well as repression of the accumulation of protochlorophyllide, a phototoxic intermediate of chlorophyll synthesis. Therefore, comparation of the Wt and ein3-1eil1-1 mutant etiolated seedlings' gene expression at genome wide will be helpfull for us to find the EIN3/EIL1 target genes in chlorophyll biosynthesis pathway. Here we used microarrays to detail the global gene expression under the regulation of EIN3/EIL1.Here we used microarrays to detail the global gene expression under the regulation of EIN3/EIL1.