Dataset Information

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channnel SLAH3

ABSTRACT: Analysis of Arabidopsis nanodomains shows that Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channnel SLAH3. The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin AtRem 1.3. After transient coexpression, CPK21 interacted with SLAH3 (SLAC1 homolog 3) and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21. Data processing and bioinformatics: MS result peak files were run on a Mascot daemon using the Mascot algorithm (version 2.2; Matrix Science) with TAIR v9 protein database, trypsin/P as protease. Allowed fixed modification was carbamidomethylation (C), and variable modifications were oxidized methionines (N) and pyroglutamic acid (pyro-Glu at N-terminal Q). Peptide and fragment mass tolerance were set to +-1.5 Da for the ion trap and +-0.2 Da for the Quad-TOF, maximum missed cleavages to two, and only singly, doubly, and triply charged ions were analyzed. After manual inspection, protein identifications with at least three unique identified peptides were automatically approved. Protein identifications based on two unique identified were only regarded if the two unique identified peptides displayed an ions score above the Mascot significance threshold for P < 0.05 (Mascot score >40 for the ion trap and >32 for the QUAD-TOF). Detailed information for the identified proteins was queried from the Uniprot consortium database (www.uniprot.org) and The Arabidopsis Information Center (TAIR: www.arabidopsis.org). The functional classification was assigned manually according to the annotations found in these two databases. Two independent biological replicates were analyzed: first setup of DRM isolation was performed with the detergents TritonX-100 and Brij-98 in-solution and in-gel digested. The second setup for DRM isolation was only performed with the detergent Triton X-100 and in-gel digested but subjected to methylcyclodextrin treatment to identify sterol-dependent lipid nanodomain proteins. For the proteomic analysis of DRMs data sets from both experimental setups were analyzed; the MCD treatment was only analyzed for the second setup. Protein amounts were estimated only for the MCD treatment using the Exponentially Modified Protein Abundance Index, which correlates with the number of observed and observable peptides per protein.

INSTRUMENT(S): LCQ Deca XP Plus, QSTAR

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Yvonne Reinders

LAB HEAD: Yvonne Reinders

PROVIDER: PXD000211 | Pride | 2013-05-02

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 431

Publications

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

Demir Fatih F Horntrich Claudia C Blachutzik Jörg O JO Scherzer Sönke S Reinders Yvonne Y Kierszniowska Sylwia S Schulze Waltraud X WX Harms Gregory S GS Hedrich Rainer R Geiger Dietmar D Kreuzer Ines I

Proceedings of the National Academy of Sciences of the United States of America 20130429 20

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cel ...[more]

PMID: 23630285

Similar Datasets

Project description:Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts. Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts. Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts.

Project description:Summary: The non-protein amino acid beta-aminobutyric acid (BABA) primes Arabidopsis to respond more quickly and strongly to pathogen and osmotic stress. Here, we report that BABA also significantly enhanced acquired thermotolerance in Arabidopsis. This thermotolerance was dependent on heat shock protein 101, a critical component of the normal heat shock response. BABA did not enhance basal thermotolerance under a severe heat shock treatment. No roles for the hormones ethylene and salicylic acid in BABA-induced acquired thermotolerance were identified by mutant analysis. By global gene expression analysis, transcript levels for several transcription factors and DNA binding proteins regulating responses to the stress hormone abscisic acid (ABA) were found to be elevated in BABA-treated relative to water-treated plants. The role of ABA in BABA-induced thermotolerance was complex. BABA-enhanced thermotolerance was partially compromised in the ABA-insensitive mutant, abi1-1, but was augmented in abi2-1. In an unrelated process, BABA, like ABA, inhibited root growth and the level of inhibition was roughly additive in roots treated with both compounds. Root growth of both abi1-1 and abi2-1 was also inhibited by BABA. Unexpectedly, abi1-1 and abi2-1 root growth was inhibited more strongly by combined ABA and BABA treatments than by BABA alone. Our results together with previously published data suggest that BABA is a general enhancer of plant stress resistance and that cross-talk occurs between BABA and ABA signalling cascades. Specifically, the BABA-mediated accumulation of ABA transcription factors without concomitant activation of a downstream ABA response could represent one component of the BABA-primed state in Arabidopsis. Overall design: A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Arabidopsis Col-0 plants were grown in ProMix HP (Premier Horticulture, Red Hill, PA, USA) for 14 days and the soil was drenched with a 0.25 mM BABA solution 1 day prior to harvesting samples for RNA preparation. These plants were grown in a growth chamber at 21?C with a 14-hr photoperiod. All plants were grown at a light intensity of about 100 ? 150 ?E m-2 s-1. BABA (mixed isomers, Sigma, Milwaukee, WI, USA) was diluted in water at indicated concentration. A biological sample represents a comparison between plants that were water-treated (green channel) to plants drenched with BABA (red channel). This study represents 4 independent biological replicates. Computed

			Action	DRS
	090612_01.wiff	Wiff
	090612_02.wiff	Wiff
	090612_03.wiff	Wiff
	090612_04.wiff	Wiff
	090612_05.wiff	Wiff