Project description:We report that CBP20 phosphorylation can regulate root growth in ethylene. We examined the gene expression in roots and shoots of wild type (Col) and cbp20 mutant (in Col background). Ethylene is one of the most essential hormones for plant developmental processes and stress responses. EIN2 is a key factor in ethylene signaling pathway and its function is regulated by phosphorylation. However, the phosphorylation regulation in the ethylene signaling pathway is largely unknown. Here we report the phosphorylation of CBP20 is regulated by ethylene, and the phosphorylation is involved in root elongation. The constitutive phosphorylation format of CBP20 rescues the cbp20 root ethylene hyposensitive phenotype, while the constitutive de-phosphorylation format of CBP20 is unable to rescue the root phenotype of cbp20 in response to ethylene. Genome wide study on ethylene regulated gene expression and microRNA expression in the roots and shoots of both Col and cbp20, together with the result of genetics validation suggest that ethylene induced phosphorylation of CBP20 is involved in root growth and one pathway is through the regulation of microRNAs and their target genes in roots.
Project description:We report that CBP20 phosphorylation can regulate root growth in ethylene. We examined the small RNA expression in roots and shoots of wild type (Col) and cbp20 mutant (in Col background). Ethylene is one of the most essential hormones for plant developmental processes and stress responses. EIN2 is a key factor in ethylene signaling pathway and its function is regulated by phosphorylation. However, the phosphorylation regulation in the ethylene signaling pathway is largely unknown. Here we report the phosphorylation of CBP20 is regulated by ethylene, and the phosphorylation is involved in root elongation. The constitutive phosphorylation format of CBP20 rescues the cbp20 root ethylene hyposensitive phenotype, while the constitutive de-phosphorylation format of CBP20 is unable to rescue the root phenotype of cbp20 in response to ethylene. Genome wide study on ethylene regulated gene expression and microRNA expression in the roots and shoots of both Col and cbp20, together with the result of genetics validation suggest that ethylene induced phosphorylation of CBP20 is involved in root growth and one pathway is through the regulation of microRNAs and their target genes in roots.
Project description:Ethylene is one of the most important hormones for plant developmental processes and stress responses. However, the phosphorylation regulation in the ethylene signaling pathway is largely unknown. Here we report the phosphorylation of cap binding protein 20 (CBP20) at Ser245 is regulated by ethylene, and the phosphorylation is involved in root growth. The constitutive phosphorylation mimic form of CBP20 (CBP20S245E or CBP20S245D), while not the constitutive de-phosphorylation form of CBP20 (CBP20S245A) is able to rescue the root ethylene responsive phenotype of cbp20. By genome wide study with ethylene regulated gene expression and microRNA (miRNA) expression in the roots and shoots of both Col-0 and cbp20, we found miR319b is up regulated in roots while not in shoots, and its target MYB33 is specifically down regulated in roots with ethylene treatment. We described both the phenotypic and molecular consequences of transgenic over-expression of miR319b. Increased levels of miR319b (miR319bOE) leads to enhanced ethylene responsive root phenotype and reduction of MYB33 transcription level in roots; over expression of MYB33, which carrying mutated miR319b target site (mMYB33) in miR319bOE is able to recover both the root phenotype and the expression level of MYB33. Taken together, we proposed that ethylene regulated phosphorylation of CBP20 is involved in the root growth and one pathway is through the regulation of miR319b and its target MYB33 in roots.
Project description:Ethylene gas is essential for many developmental processes and stress responses in plants. ETHYLENE INSENSITIVE2 (EIN2), an NRAMP-homologous integral membrane protein, plays an essential role in ethylene signaling but its function remains enigmatic. Here we report that phosphorylation-regulated proteolytic processing of EIN2 triggers its endoplasmic reticulum (ER)-nucleus translocation, which is essential for hormone signaling and response in Arabidopsis. Without ethylene, or in hormone receptors mutants, ER-tethered EIN2 shows CTR1 kinase-dependent phosphorylation. Ethylene exposure triggers dephosphorylation and proteolytic cleavage, resulting in rapid nuclear translocation of a carboxyl-terminal EIN2 fragment (C’). Plants containing mutations that mimic EIN2 dephosphorylation, or inactivate CTR1, show constitutive cleavage and nuclear localization of EIN2-C’, and EIN3/EIL1-dependent activation of ethylene responses. These findings uncover a mechanism of subcellular communication whereby ethylene gas stimulates rapid phosphorylation-dependent cleavage and nuclear movement of the EIN2-C’ peptide, thus linking hormone perception and signaling components located in the ER with nuclear-localized transcriptional regulators.
Project description:Senescence represents the last stage of flower development. Phosphorylation is one of the key post-translational modifications that regulate protein functions in diverse biological pathways and contexts. Generally, kinases may be more required than phosphatases during plant growth and development. However, little is known about global phosphorylation change during flower senescence. In this work, we quantitatively investigated the petunia phosphoproteome following ethylene or air treatment. In total, 2170 phosphosites in 1184 protein groups were identified, among which 2059 sites in 1124 proteins were quantified. Treatment with ethylene resulted in 711 down-regulated and only 117 up-regulated phosphosites using a 1.5-fold threshold (P<0.05), showing that ethylene negatively regulates global phosphorylation levels and that phosphorylation of lots of proteins was not necessary during flower senescence. Our results show that protein dephosphorylation may play an important role of in ethylene-induced corolla senescence in petunia and that phosphatases may be more required than kinases during flower senescence. In addition, our results show that ethylene regulates ethylene and ABA signaling transduction pathways via phosphorylation level, and plant mRNA splicing machinery was a major target of ethylene-induced dephosphorylation. Moreover, ethylene treatment increases the number of alternative splicing of precursor RNAs in petunia corollas.
Project description:Despite their importance, plant MAP kinase targets are still poorly elucidated. Here, the specific in vivo interaction of an ethylene response factor (ERF104) with the Arabidopsis MAP kinase, MPK6, is shown by fluorescence resonance energy transfer. The interaction, which is lost within minutes after treatment with the flagellin-derived flg22 peptide, is dependent on both MPK6 kinase activity and rapid ethylene signaling initiated downstream of MPK6 activation. ERF104 is an MPK6 substrate and phosphorylation site mutations affected its stability. ERF104 activates promoters with GCC elements. This was evident from microarray data of overexpressing transgenic plants, where promoters of up regulated genes contain GCC motifs and chromatin immunoprecipitation showing ERF104 association with PDF1.2 promoter. The ERF104 overexpressor did not affect biotrophic bacteria proliferation but was more susceptible to necrotrophic Botrytis cinerea. Microarray performed with erf104 or mpk6 revealed only a limited number of flg22-induced genes that require these elements - possibly as a result of functional redundancies. Thus, ERF104 phosphorylation by MPK6, in concert with ethylene signaling induced by pathogen-derived molecules, modulates defense in Arabidopsis.
Project description:Ethylene-dependent gene expression was assayed by treating with 1 uM ACC, an ethylene precursor, or a control treatment to Arabidopsis seedlings by transferring 6 day old Arabidopsis grown on a a nylon mesh to fresh ACC-containing or control media Seedling roots were harvested 0, .5, 1, 2, 4, 8, 12, and 24 hours after treatment and the resultant RNA was used for microarray analysis to determine the kinetic profiles of auxin-responsive gene expression
Project description:Ethylene induced hyponastic growth in Arabidopsis thaliana F.F. Millenaar L.A.C.J. Voesenek and A.J.M. Peeters Our aim is to identify genes involved in the ethylene induced hyponastic growth. Upon submergence some plant species like Rumex palustris changes its leaf angle (hyponastic growth) and shows enhanced petiole elongation to reach the water surface. In Rumex palustris the hyponastic growth is initiated by an increased concentration of ethylene due to physical entrapment and ongoing ethylene biosynthesis. A proteomics, genomics and genetical approach to improve our understanding of above described flooding-induced responses are not feasible in Rumex palustris since genomic information about this species is limited. However it is possible to use the model plant Arabidopsis thaliana as a tool in flooding research. Natural accessions (Be0 Col Cvi Kas Ler Nd Rld Shah and Ws) show considerable genetic variation in hyponastic growth upon exposure to ethylene Col exhibiting the largest effect (maximum rate after 3 hours) and Ler no effect whatsoever. Using a computer controlled digital camera the hyponastic growth is measured in great detail. Next to ethylene addition also a transfer to low light causes hyponastic growth. This seems to be an ethylene independent pathway because etr1 and ctr1 showed hyponastic growth after transfer to low light. Ethylene and low light showed additive effects in Col. It is likely that ethylene induces more changes in gene expression than only the ones involved in hyponastic growth. By subtracting changes in the Ler expression profile from changes in the Col expression profile we expect to find why Col and Ler respond differently on ethylene by finding specific ethylene induced genes that are involved in hyponastic growth. The expression profile of Col following transfer to low light will be substracted from Col following ethylene addition to distinguish between genes that are involved in hyponastic growth but are not specific for ethylene induced hyponastic growth. There are strong indications in Rumex palustris that other hormones i.e. auxin ABAand GA are involved in the ethylene induced hyponastic growth. Currently mutants in ethylene auxin and ABA biosynthesis and/or signal transduction are screened for hyponastic growth. Preliminary results showed that also in Arabidopsis these other hormones are involved in ethylene induced hyponastic growth. Beside the mutant approach we also started a proteomics and a PCR based differential screen approach. Together with the proposed transcriptome analysis we hope to find new genes involved in ethylene induced hyponastic growth.