Dataset Information

Novel TTG1 Mutants Modify Root-Hair Pattern Formation in Arabidopsis.

ABSTRACT: The patterning of root-hair and non-hair epidermal cells in the Arabidopsis root is governed by a network of transcriptional regulators. The central MYB-bHLH-WD40 (MBW) transcriptional complex includes the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). To clarify the role of TTG1, we describe the identification and analysis of two new ttg1 mutants. Each of these mutants contains a single nucleotide change in the TTG1 gene, which causes a single amino-acid substitution in the predicted TTG1 protein and alters root-hair pattern formation. Surprisingly, these new ttg1 mutants exhibit decreased root-hair formation, particularly in the caprice (cpc) mutant background, rather than increased root-hair formation as reported for strong ttg1 mutants. We show that the unique phenotype of these mutants is due to differential effects of the altered TTG1 proteins on target gene expression, associated with a weakened ability to interact with its GLABRA3 bHLH partner. These findings demonstrate the crucial role of TTG1 for the appropriate balance of target gene activation to achieve the proper pattern of epidermal cell types during Arabidopsis root development.

SUBMITTER: Long Y

PROVIDER: S-EPMC7154166 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Novel TTG1 Mutants Modify Root-Hair Pattern Formation in Arabidopsis.

Long Yun Y Schiefelbein John J

Frontiers in plant science 20200407

The patterning of root-hair and non-hair epidermal cells in the Arabidopsis root is governed by a network of transcriptional regulators. The central MYB-bHLH-WD40 (MBW) transcriptional complex includes the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). To clarify the role of TTG1, we describe the identification and analysis of two new ttg1 mutants. Each of these mutants contains a single nucleotide change in the TTG1 gene, which causes a single amino-acid substitution in the ...[more]

PMID: 32318087

Similar Datasets

Project description:Using cell sorting technique, we collected Arabidopsis root hair protoplasts (transgenic line harboring root hair specific gene promoter ::GFP, here named GFP) and nonGFP protoplasts( whole root except root hair). Then, we extracted total proteins and RNA for proteome and RNASeq. RNAseq data have already been deposited into NCBI (accession numbers SRR364677 and SRR364678). Total proteins were separated by 1D SDS-PAGE, followed by gel slicing, in gel digested and run ESI-LC-MS/MS on LTQ Orbitrap Velos. We set two biological repeats and each sample was scanned two times (two technique repeats). We then combined all ms/ms raw data from all fractions and technique repeats from one biological sample into one file to indentify proteins by searching Arabidopsis protein database with software Proteome Discoverer (with two searching engines mascot and sequester). Protocol: Protoplasts from EXP7 and non-GFP protoplastswere stored at -80°C, thawed on ice for 5 min, vortexed several times and suspended in 5 x volume of pre-cooled acetone (-20°C) containing 10% (v/v) trichloroacetic acid (TCA) and 0.07% (v/v) 2-mercaptoethanol. Proteins were then precipitated for 2 h at −20°C after thorough mixing. Proteins were collected by centrifuging at 35,000 g (JA-20 108 rotor, Beckman J2-HS) at 4°C for 30 min. The supernatant was removed and the protein pellets were washed three times with cold acetone containing 0.07% (v/v) 2-mercaptoethanol and 1 mMphenylmethanesulfonylfluoride. Protein pellets were dried by lyophilization and stored at -80°C, or immediately extracted using Laemmli buffer (63 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue). Protein concentration was determined using Pierce protein assay kit (Pierce, Rockford). Proteins from each sample were separated using a Bis-Tris precast gradient gel (4−16%, Bio-Rad) according to the manufacturer’s instructions. After electrophoresis, the gel was Coomassie-stained using the Colloidal Blue Staining Kit (Invitrogen). The protocol used for in-gel trypsin digestion of proteins in gels was adapted from the method described in Shevchenko et al. (1996). Briefly, protein bands were manually excised and each band was cut into small pieces (~0.5 mm). The gel pieces were washed three times with a solution containing 50% methanol and 5% acetic acid for 2 h, twice with a solution containing 25 mM NH4HCO3 in 50% acetonitrile for 10 min each, and then the gel pieces were dried in a vacuum centrifuge. Reduction of proteins in gel pieces was performed with DTT and alkylation with iodoacetamide, and the gel pieces were washed and dried in a vacuum centrifuge. A trypsin solution in 25 mM NH4HCO3 containing 75 to 100 ng of sequencing grade modified trypsin (Promega) in 25-40 µl was added and incubated with gel pieces for 12-16 h at 37°C. To recover the tryptic peptides, a solution of 30 µl containing 5% formic acid and 50% acetonitril was added to the gel pieces, agitated in a vortex for 30-60 min and withdrawn into a new tube. The recovery was repeated once with 15 µl solution and the resulting two recovers were combined and dried in a vacuum centrifuge. The dried pellet was re-dissolved in 10-20 µl of 0.1% formic acid for LC-MS/MS analysis.

Dataset Information

Novel TTG1 Mutants Modify Root-Hair Pattern Formation in Arabidopsis.

Publications

Novel <i>TTG1</i> Mutants Modify Root-Hair Pattern Formation in Arabidopsis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets