Anders Laurell Blom Møller

Specific Aquaporins Facilitate the Diffusion of Hydrogen Peroxide across Membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

Aquaporin homologues in plants and mammals transport ammonia

Using functional complementation and a yeast mutant deficient in ammonium (NH4 +) transport (Δmep1–3), three wheat (Triticum aestivum) TIP2 aquaporin homologues were isolated that restored the ability of the mutant to grow when 2 mM NH4 + was supplied as the sole nitrogen source. When expressed in Xenopus oocytes, TaTIP2;1 increased the uptake of NH4 + analogues methylammonium and formamide. Furthermore, expression of TaTIP2;1 increased acidification of the oocyte‐bathing medium containing NH4 + in accordance with NH3 diffusion through the aquaporin. Homology modeling of TaTIP2;1 in combination with site directed mutagenesis suggested a new subgroup of NH3‐transporting aquaporins here called aquaammoniaporins. Mammalian AQP8 sharing the aquaammoniaporin signature also complemented NH4 + transport deficiency in yeast.

Responses of barley root and shoot proteomes to long-term nitrogen deficiency, short-term nitrogen starvation and ammonium.

Cereals are major crops worldwide, and improvement of their nitrogen use efficiency is a crucial challenge. In this study proteins responding to N supply in barley roots and shoots were analysed using a proteomics approach, to provide insight into mechanisms of N uptake and assimilation. Control plants grown hydroponically for 33 d with 5 mm nitrate, plants grown under N deficiency (0.5 mm nitrate, 33 d) or short-term N starvation (28 d with 5 mm nitrate followed by 5 d with no N source) were compared. N deficiency caused changes in C and N metabolism and ascorbate-glutathione cycle enzymes in shoots and roots. N starvation altered proteins of amino acid metabolism in roots. Both treatments caused proteome changes in roots that could affect growth. Shoots of plants grown with ammonium as N source (28 d with 5 mm nitrate followed by 5 d with 5 mm ammonium) showed responses similar to N deficient shoots, characterized by turnover of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and increases in proteins of the chloroplastic transcription and translation machinery. Identified proteins in 67 and 49 varying spots in roots and shoots respectively, corresponded to 62 functions and over 80 gene products, considerably advancing knowledge of N responses in barley.

A workflow for peptide-based proteomics in a poorly sequenced plant: A case study on the plasma membrane proteome of banana

Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.

New approach for segmentation and quantification of two-dimensional gel electrophoresis images

MOTIVATION Detection of protein spots in two-dimensional gel electrophoresis images (2-DE) is a very complex task and current approaches addressing this problem still suffer from significant shortcomings. When quantifying a spot, most of the current software applications include a lot of background due to poor segmentation. Other software applications use a fixed window for this task, resulting in omission of part of the protein spot, or including background in the quantification. The approach presented here for the segmentation and quantification of 2-DE aims to minimize these problems. RESULTS Five sections from different gels are used to test the performance of the presented method concerning the detection of protein spots, and three gel sections are used to test the quantification of sixty protein spots. Comparisons with a state-of-the-art commercial software and an academic state-of-the-art approach are presented. It is shown that the proposed approach for segmentation and quantification of 2-DE images can compete with the available commercial and academic software packages. AVAILABILITY A command-line prototype may be downloaded, for non-commercial use, from http://w3.ualg.pt/~aanjos/prototypes.html.

Plasma membrane proteome analysis identifies a role of barley Membrane Steroid Binding Protein in root architecture response to salinity

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity.

Plant Plasma Membrane Proteomics: Challenges and Possibilities

Plasma membrane proteins are challenging to study due to their hydrophobicity and low abundance. Extraction of plasma membranes from plants is complicated by the rigid plant cell wall. The most commonly used method to enrich for plant plasma membranes is the aqueous-polymer two-phase system. Many gel-based separation techniques have been used in plasma membrane proteomics studies including one-dimensional SDS-PAGE, classical two-dimensional electrophoresis based on isoelectric focussing and SDS-PAGE, double SDS-PAGE, Blue Native-PAGE and 16-BAC-PAGE. Some of the difficulties associated with gel-based separation of hydrophobic proteins can be bypassed by the use of gel-free approaches which involve in-solution separation of proteins or liquid chromatography-based separation of peptides. Mass spectrometry is subsequently used for identification of proteins. An overview is presented of the most commonly used methods for analysis of plant plasma membrane proteomes, their strengths and weaknesses.