Mapping and proteomic analysis of albumin and globulin proteins in hexaploid wheat kernels (Triticum aestivum L.)

Albumins and globulins of wheat endosperm represent 20% of total kernel protein. They are soluble proteins, mainly enzymes and proteins involved in cell functions. Two-dimensional gel immobiline electrophoresis (2DE) (pH 4-7) × SDS-Page revealed around 2,250 spots. Ninety percent of the spots were common between the very distantly related cultivars ‘Opata 85’ and ‘Synthetic W7984’, the two parents of the International Triticeae Mapping Initiative (ITMI) progeny. ‘Opata’ had 130 specific spots while ‘Synthetic’ had 96. 2DE and image analysis of the soluble proteins present in 112 recombinant inbred lines of the F9-mapped ITMI progeny enabled 120 unbiased segregating spots to be mapped on 21 wheat (Triticum aestivum L. em. Thell) chromosomes. After trypsic digestion, mapped spots were subjected to MALDI-Tof or tandem mass spectrometry for protein identification by database mining. Among the ‘Opata’ and ‘Synthetic’ spots identified, many enzymes have already been mapped in the barley and rice genomes. Multigene families of Heat Shock Proteins, beta-amylases, UDP-glucose pyrophosphorylases, peroxydases and thioredoxins were successfully identified. Although other proteins remain to be identified, some differences were found in the number of segregating proteins involved in response to stress: 11 proteins found in the modern selected cultivar ‘Opata 85’ as compared to 4 in the new hexaploid `Synthetic W7984’. In addition, ‘Opata’ and ‘Synthetic’ differed in the number of proteins involved in protein folding (2 and 10, respectively). The usefulness of the mapped enzymes for future research on seed composition and characteristics is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fermentation pH Influences the Physiological-State Dynamics of Lactobacillus bulgaricus CFL1 during pH-Controlled Culture

This study aims at better understanding the effects of fermentation pH and harvesting time on Lactobacillus bulgaricus CFL1 cellular state in order to improve knowledge of the dynamics of the physiological state and to better manage starter production. The Cinac system and multiparametric flow cytometry were used to characterize and compare the progress of the physiological events that occurred during pH 6 and pH 5 controlled cultures. Acidification activity, membrane damage, enzymatic activity, cellular depolarization, intracellular pH, and pH gradient were determined and compared during growing conditions. Strong differences in the time course of viability, membrane integrity, and acidification activity were displayed between pH 6 and pH 5 cultures. As a main result, the pH 5 control during fermentation allowed the cells to maintain a more robust physiological state, with high viability and stable acidification activity throughout growth, in opposition to a viability decrease and fluctuation of activity at pH 6. This result was mainly explained by differences in lactate concentration in the culture medium and in pH gradient value. The elevated content of the ionic lactate form at high pH values damaged membrane integrity that led to a viability decrease. In contrast, the high pH gradient observed throughout pH 5 cultures was associated with an increased energetic level that helped the cells maintain their physiological state. Such results may benefit industrial starter producers and fermented-product manufacturers by allowing them to better control the quality of their starters, before freezing or before using them for food fermentation.Lactic acid bacteria are traditionally used to produce or to preserve various food products such as fermented milks, meats, and vegetables. Their ability to initiate rapid acidification of the raw material is essential to improve the flavor, texture, and safety of these products (11, 14). In order to prevent poor fermentation yields and to improve the quality and reliability of the products, it is important to maintain proper control starter production. This control may be achieved by studying the effects of process parameters on the growth kinetics of the bacteria and on their acidification activity and physiological state in growing conditions. Among all process parameters, pH and harvesting time are key factors that strongly influence the physiological state of lactic acid bacteria after fermentation and stabilization.Lactic acid starters are currently produced using pH-controlled pure cultures (6), during which pH is generally regulated at an optimal value by continuously adding sodium hydroxide or ammonia in the bioreactor (23). Various growth characteristics such as maximal biomass concentration, specific growth rate, fermentation time, sugar consumption or growth, and product yields are significantly influenced by the pH control value (1, 4). Optimal pH ranges were therefore determined for several lactic acid bacteria, such as Streptococcus thermophilus (pH 6.5), Lactobacillus bulgaricus (pH 5.8 to 6) (5, 22), or Lactococcus lactis subsp. cremoris (pH 6.3 to 6.9) (8).Compared to acidic fermentations, pH-controlled cultures led to higher growth yields and productivity (9, 23) as a result of the lower level of nondissociated lactic acid in the culture medium (2, 12, 15). The acidification of the cytoplasm induced by the nondissociated form of the weak organic acid leads to the collapse of the proton motive force (13). This phenomenon inhibits nutrient transport and enzymatic reactions and leads to DNA alteration and biomass inactivation (12). Maintaining the extracellular pH (pHext) at a high value helps the cells stabilize their intracellular pH at a sufficiently high value (9), thus decreasing the inhibiting effect of lactic acid.Fermentation pH also acts on energetic parameters, such as internal pH (pHi), pH gradient (dpH), proton motive force, membrane potential, NADH/NAD ratio, ATP level and rate of ATP formation, and lactate dehydrogenase and ATPase activity (1, 9, 17). During batch cultures of L. lactis performed with or without pH control, Cachon et al. (9) showed that pH control has a significant influence on the variations of pHi, dpH, and NADH/NAD ratio, thus acting on growth parameters. Moreover, in batch cultures, pHi is dependent upon both the external pH and the age of culture. Mercade et al. (17) showed that cultures of L. bulgaricus at controlled pH 6.4 are inhibited at the level of anabolism but were not energy limited. They are characterized by a high maintenance coefficient in contrast to cultures without pH control which consume intracellular energy for pHi regulation.The effect of pH on cellular physiology is confirmed by other studies which show that it influences acidification activity of lactic acid bacteria (23-25). Whereas Wang et al. (25) indicated that Lactobacillus acidophilus cells grown at optimal pH display a higher residual acidification activity than cells grown at lower pH control values, Schepers et al. (24) and Savoie et al. (23) demonstrated that this activity is higher when starters are produced without pH control or at low pH control values. These authors explained that conditions generating high biomass concentrations do not systematically lead to cells with an efficient acidification activity.From this information, the effect of pH control was elucidated on growth and energetic parameters, whereas its effect on the dynamic of cellular physiology, viability, and acidification activity during growth is still not determined.A few authors demonstrated that the harvesting time has a strong impact on cellular parameters such as viability and acidification activity (3, 20, 24). Béal et al. (6) specified that there is an optimal range of time during which to harvest cells in a good physiological state, i.e., at a high cellular concentration and a high acidification activity. However, since this optimal range is strongly strain and condition dependent, more information is needed about the influence of harvesting time on physiological parameters.In order to improve knowledge about the effects of fermentation pH and harvesting time on starter''s quality, we sought here to apply some rapid and relevant methods to characterize the dynamic of L. delbrueckii subsp. bulgaricus CFL1 physiological state throughout pH 6 and pH 5 fermentations. This might allow industrial starter producers to better control their fermentations and to achieve high-quality starters. Among the available methods, the Cinac system and multiparametric flow cytometry, associated with plate counts, made it possible to determine and compare different physiological parameters such as cultivability, acidification activity (Cinac system), membrane damage, enzymatic activity, cell depolarization, intracellular pH, and pH gradient (flow cytometry) (20). Two dynamic schemes of the time course of the physiological state during pH 6 or pH 5 cultures are proposed and discussed.     

Coevolution between a Family of Parasite Virulence Effectors and a Class of LINE-1 Retrotransposons

Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVR _k1 powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVR _k1 gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome.     

Where and how Argentine ant (Linepithema humile) spreads in Corsica?

The Argentine ant, Linepithema humile (Dolichoderinae), is one of the most widespread invasive ant species in the world. When established in optimal habitat, this species usually excludes most other local ants and can heavily impact other arthropods as well. Although Argentine ants have been present in southern Europe for more than 100 years, they were first noted in Corsica, a French Mediterranean island, in 1957 in only one urban station. In this study, we aimed to map precisely their geographical distribution in Corsica and to quantify their presence by using an infestation index. We recorded changes in the distribution of Argentine ants in Corsica over the past decade. Argentine ants appeared to be well established within their introduced range and spreading along the Corsican coasts principally through Human-mediated jump-dispersal but not homogenously. To cite this article: O. Blight et al., C. R. Biologies 332 (2009).     

Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.     

T cells in ANCA-associated vasculitis: what can we learn from lesional versus circulating T cells?

Anti-neutrophil cytoplasmic antibody (ANCA) - associated vasculitis (AAV) is a life-threatening autoimmune disease characterized by an antibody-mediated glomerulonephritis and necrotizing vasculitis. Apart from antibodies, T cells are also involved in disease pathogenesis. This review stresses the hallmarks of T cell-mediated pathology in AAV and highlights the characteristics of lesional and circulating T cells in the immune response in AAV. Circulating effector T-cell populations are expanded and are in a persistent state of activation. Circulating regulatory T-cell subsets are less well characterized but seem to be impaired in function. Lesional effector T cells are present in granulomas, vasculitic lesions, and nephritis. Lesional T cells usually show pro-inflammatory properties and promote granuloma formation. Apart from T cells, dendritic cells are abundantly present at the sites of inflammation and locally orchestrate the immune response. Targeting the above-mentioned T cell-mediated disease mechanisms will potentially provide powerful therapeutic tools for AAV.     

O-Mycoloylated Proteins from Corynebacterium: AN UNPRECEDENTED POST-TRANSLATIONAL MODIFICATION IN BACTERIA*

O-Acylation of proteins was known only in a few eukaryotic proteins but never in bacteria. We demonstrate, using a combination of protein chemistry and mass spectrometry, the occurrence of three O-acylated polypeptides in Corynebacterium glutamicum, PorA, PorH, and an unknown small protein. The three polypeptides are O-substituted by mycolic acids, long chain α-alkyl and β-hydroxy fatty acids specifically produced by members of the Corynebacterineae suborder. To date these acids were described only as esterifying trehalose and arabinogalactan, and less frequently glycerol, important components of the highly impermeable outer barrier of Corynebacterineae. We show that the post-translational mycoloylation of PorA occurs at Ser-15 and is necessary for the pore-forming activity of C. glutamicum.     

A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens exhibits a unique capacity for cadmium tolerance and accumulation. We investigated the molecular basis of this exceptional Cd(2+) tolerance by screening for T. caerulescens genes, which alleviate Cd(2+) toxicity upon expression in Saccharomyces cerevisiae. This allowed for the isolation of a cDNA encoding a peptide with homology to the C-terminal part of a heavy metal ATPase. The corresponding TcHMA4 full-length sequence was isolated from T. caerulescens and compared to its homolog from Arabidopsis thaliana (AtHMA4). Expression of TcHMA4 and AtHMA4 cDNAs conferred Cd sensitivity in yeast, while expression of TcHMA4-C and AtHMA4-C cDNAs encoding the C-termini of, respectively, TcHMA4 and AtHMA4 conferred Cd tolerance. Moreover, heterologous expression in yeast suggested a higher Cd binding capacity of TcHMA4-C compared to AtHMA4-C. In planta, both HMA4 genes were expressed at a higher level in roots than in shoots. However, TcHMA4 shows a much higher constitutive expression than AtHMA4. Our data indicate that HMA4 could be involved in Cd(2+) transport and possibly in the Cd hyperaccumulation character.