Using phosphoproteomics to reveal signalling dynamics in plants

To ensure appropriate responses to stimuli, organisms have evolved signalling networks that rely on post-translational modifications of their components. Among these, protein phosphorylation has a prominent role and much research in plants has focused on protein kinases and phosphatases, which, respectively, catalyse phosphorylation and dephosphorylation of specific substrates. Technical limitations, however, have hampered the identification of these substrates. As reviewed here, novel mass spectrometry-based techniques have enabled the large-scale mapping of in vivo phosphorylation sites. Alternatively, methods based on peptide and protein microarrays have revealed protein kinase activities in cell extracts, in addition to kinase substrates. A combined phosphoproteomic approach of mass spectrometry and microarray technology could enhance the construction of dynamic plant signalling networks that underlie plant biology.     

The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control

Programmed cell death (PCD) is a common host response to microbial infection [1-3]. In plants, PCD is associated with immunity to biotrophic pathogens, but it can also promote disease upon infection by necrotrophic pathogens [4]. Therefore, plant cell-suicide programs must be strictly controlled. Here we demonstrate that the Arabidopsis thaliana Brassinosteroid Insensitive 1 (BRI1)-associated receptor Kinase 1 (BAK1), which operates as a coreceptor of BRI1 in brassinolide (BL)-dependent plant development, also regulates the containment of microbial infection-induced cell death. BAK1-deficient plants develop spreading necrosis upon infection. This is accompanied by production of reactive oxygen intermediates and results in enhanced susceptibility to necrotrophic fungal pathogens. The exogenous application of BL rescues growth defects of bak1 mutants but fails to restore immunity to fungal infection. Moreover, BL-insensitive and -deficient mutants do not exhibit spreading necrosis or enhanced susceptibility to fungal infections. Together, these findings suggest that plant steroid-hormone signaling is dispensable for the containment of infection-induced PCD. We propose a novel, BL-independent function of BAK1 in plant cell-death control that is distinct from its BL-dependent role in plant development.     

Strategies for recycling and valorization of grape marc

Grapes are one of the most cultivated fruit crops worldwide. Either for wine or juice production, grape processing generates a large amount of residues that must be treated, disposed of or reused properly to reduce their pollution load before being applied to the soil. In this review, a special focus is given to the treatment and valorization of the winemaking by-product like grape marc via anaerobic digestion, composting and vermicomposting at laboratory, pilot, and industrial scales. The impact of the final products (digestates, composts, and vermicomposts) on soil properties is briefly addressed. Moreover, the role of grape marc and seeds as a valuable source of natural phytochemicals that include polyphenols and other bioactive compounds of interest for pharmaceutical, cosmetic, and food industries is also discussed. This is of paramount importance given the fact that sustainability requires the use of management and valorization strategies that allow the recovery of valuable compounds (e.g. antioxidants) with minimum disposal of waste streams.     

Cross-reactivity of monoclonal antibodies against phytochrome from Zea and Avena

The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Avena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg · mol^-1 phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120–125 kg·mol^-1 from several ferns, a liverwort and mosses. This antibody (Z-3B1), which was localized within a 23.5-kg·mol^-1 section of Avena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical.Abbreviations ELISA enzyme-linked immunosorbent assay - McAb monoclonal antibody - PBS phosphate-buffered saline - Pfr (Pr) far-red-absorbing (red-absorbing) form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The axonally secreted cell adhesion molecule, axonin-1. Primary structure, immunoglobulin-like and fibronectin-type-III-like domains and glycosyl-phosphatidylinositol anchorage.

Axonin-1 is an axon-associated cell adhesion molecule (AxCAM) of the chicken, which promotes neurite outgrowth by interaction with the AxCAM L1(G4) of the neuritic membrane. Here we report the cloning and sequence determination of a cDNA encoding axonin-1. Peptides generated by enzymatic cleavage showed similarity to the AxCAM F11. Degenerated polymerase chain reaction (PCR) primers were designed and an axonin-1 fragment was amplified from mRNA of embryonic retina. Screening of a cDNA library from embryonic brain resulted in the isolation of a 4.0-kb cDNA insert with an open reading frame of 3108 nucleotides. The deduced polypeptide of 1036 amino acids includes a putative hydrophobic N-terminal signal sequence of 23 or 25 amino acids and a C-terminal hydrophobic sequence of 29 amino acids which is suggestive of sequences serving as signal for the attachment of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) anchor. The putative mature form of axonin-1 comprises six immunoglobulin-like repeats, followed by four fibronectin-type III repeats. Axonin-1 exhibits 75% amino acid identity with the AxCAM TAG-1 of the rat, suggesting that it is the chicken homologue of TAG-1. Like TAG-1, axonin-1 is glycosyl-PtdIns-anchored to the neuronal membrane; in contrast to TAG-1, it does not exhibit an Arg-Gly-Asp sequence.     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance