Project description:We have found that MtFTb genes play a role in the response to LD conditions, both under vernalized and non-vernalized conditions. To explore the regulatory gene network downstream of FTb genes on a global scale, we performed RNA sequencing of the gene-edited Mtftb1/b2 vs WT plants. For this, we grew wild-type and gene-edited Mtftb1/2 plants under two different conditions: vernalised long days (VLD), where the plants were vernalized for 14 days and then grown for two weeks under LD photoperiod, with the aim of capture changes in expression profiles in the period in which the plant becomes physiologically committed to flower; the second condition was non-vernalised long days (NVLD), where non-vernalized plants were grown for 60 days, a crucial time point when wild-type plants typically undergo the transition to flowering. This approach allowed us to investigate the gene expression changes in the FTb1/2 mutant independent of vernalization effects.
Project description:Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance upon environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor ANAC017, which mediates the onset of protective responses upon stress-induced mitochondrial dysfunction in Arabidopsis. Post-translational control of the elicited responses, in contrast, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B’γ, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as regulatory components induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, and that this required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and the accumulation of ACO3S91D-YFP, in turn, promoted the expression of stress related genes and ACO3 function associated with plant tolerance against UV-B or antimycin A-induced mitochondrial dysfunction. These findings positioned ACO3 both as a target and modulator of mitochondrial dysfunction signaling, critical in the attainment of stress tolerance in Arabidopsis leaves.
Project description:Non-stressed and UV-stressed IMR-32 cells were subjected to HITS-CLIP to monitor nELAVL binding changes during AD progression Nonstressed and stressed IMR-32 cells were UV-irradiated and subjected to nELAVL HITS-CLIP (detailed desription in accompanying paper)
Project description:We introduce IC-FPOMP (in-cell fast photochemical oxidation of MPs), a method enabling in situ footprinting of IMPs within live cells. IC-FPOMP generates reactive oxygen radicals from several precursors nearby the membrane. We achieve high-throughput and rapid footprinting of IMPs. IC-FPOMP of two human IMPs (human glucose transporter-hGLUT1 and human gamma glutamyl carboxylase-hGGCX) were successful, providing comprehensive footprinting of both the transmembrane and extramembrane regions. IC-FPOMP offers insights on IMP structure-function relationships in cell for the first time, facilitating drug discovery.
Project description:We have implemented the use of a heterobifunctional, UV photoactivatable cross-linker, which greatly increases the number of identified cross-links compared with homobifunctional, NHS-ester based cross-linkers. We have cross-linked human serum albumin in the context of human blood serum. We present a novel methodology that combines the use of this high-resolution cross-linking with conformational space search to investigate the structure of proteins in their native environment.
Project description:AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24°C, 50% humidity and 150 µEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups. Experimenter name = Jakub Horak , Klaus Harter; Experimenter institute = AtGenExpress Experiment Overall Design: 28 samples were used in this experiment