Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry

A method for the detection of trehalose-6-phosphate (T6P) in tissue of the model plant Arabidopsis thaliana is presented. Liquid-liquid extraction (LLE) and mixed mode solid-phase extraction (SPE) were used for sample pretreatment followed by anion exchange chromatography (AEC) coupled with electrospray ionization mass spectrometry (MS) for highly selective quantitative analysis. LLE of plant material was performed with chloroform/acetonitrile/water (3:7:16, v/v/v) followed by SPE with Oasis MAX material, which significantly reduced the complexity of the extracts. On-line coupling of MS with gradient AEC using a sodium hydroxide eluent was accomplished with a postcolumn ion suppressor. The method allows specific quantification of T6P with good linearity for spiked plant extracts, from 80 nM to 1.3 μM (r² > 0.98). The limit of detection in plant extracts was 40 nM. The recovery of the method was above 80% for relevant T6P levels. The method was applied to the determination of T6P in seedlings from four mutant A. thaliana lines (TRR1-4) resisting growth arrest caused by external supply of trehalose. Results reveal that T6P accumulation differed substantially in the four mutant lines and wild type (WT). It is concluded that the mutants circumvent the growth arrest observed in WT seedlings on 100 mM trehalose by different mechanisms.     

Silene cDNA clones for a divergent chlorophyll-a/b-binding protein and a small subunit of ribulosebisphosphate carboxylase

Summary We have isolated and analyzed cDNA clones for aSilene pratensis chlorophyll-a/b-binding protein (CAB) and a small subunit (SS) of ribulosebisphosphate carboxylase. These cDNA clones contain the coding information for the complete transit peptides. The CAB clone codes for a divergent CAB protein that differs from most published CAB sequences in both the transit peptide part and in the amino terminal part of the mature protein, a region with an important regulatory function. The SS clone codes for a precursor that is homologous to other published precursor sequences. In the mature part some non-conservative changes are observed.Silene cDNA clones for four chloroplast specific precursor proteins that are directed towards three different chloroplast compartments have been analyzed and the transit peptides compared.     

Processing of peptide precursors

Much further work will be necessary to define more fully this novel and growing mammalian family of kex2-like proteases, and future years should bring a more detailed understanding of their mechanisms and roles in precursor processing. This knowledge may also provide new insights into the mechanisms by which precursors are sorted into the appropriate intracellular organelles, correctly modified posttranslationally, processed proteolytically into their desired products, and ultimately delivered in a regulated fashion to their sites of release or action.     

Identification of a light-regulated MYB gene from an Arabidopsis transcription factor gene collection

Seven different MYB-related genes have been isolated from a genomic Arabidopsis library with probes based on MYB DNA-binding motifs. The predicted amino acid sequence of these genes showed high similarity in the MYB domain but outside this region virtually no similarities were found. The set of MYB-related genes was used to identify differentially expressed genes following the transfer of etiolated seedlings to light. This differential screen resulted in the selection of the ATM4 gene which is induced by light within one hour of exposure of etiolated or dark-adapted seedlings.     

Focus: Allergic Diseases and Type II Immunity: Model of Walnut Allergy in CC027/GeniUnc Mice Recapitulates Key Features of Human Disease

Tree nut allergies affect 1% of the United States population, are often severe in nature and rarely outgrown. Despite the severity and prevalence, there are no FDA-approved treatments for tree nut allergy. Development of a therapeutic would be expedited by having a mouse model that mimics the human disease. We utilized the CC027/GeniUnc mouse strain, which was previously identified as an orally reactive model of peanut allergy, to develop a model of walnut allergy. Mice were sensitized with walnut and cholera toxin for 4 weeks and subsequently challenged by oral gavage. Blood samples were collected to measure serum IgE. Walnut-sensitized mice produced high levels of walnut-IgE and were cross-sensitized to pecan. Oral challenges with walnut resulted in severe anaphylaxis and accompanying allergic symptoms. Importantly, pecan challenges also led to severe allergic reactions, indicating cross-reactivity to pecan. Overall, this novel mouse model reproduces key characteristics of human walnut allergy, which provides a platform to develop novel therapies and better understand sensitization mechanisms.     

Essential function in chloroplast recognition of the ferredoxin transit peptide processing region

Summary Plant ferredoxin is a nuclear-encoded chloroplast protein that is synthesized in the cytoplasm as a transit peptide-containing precursor molecule. To identify functional regions in the pre-ferredoxin transit peptide we constructed mutants with deletions of increasing length from the processing site toward the amino-terminus of the precursor. The mutant proteins were tested in an in vitro chloroplast binding and import assay. Deletion of the amino acids adjacent to the processing site completely abolishes binding and import. This region contains a sequence motif that is conserved among different precursor species. By constructing and testing mutants in the amino-terminal region of the mature part of the precursor protein, we found that this region of the molecule can greatly influence the import reaction.