Weakly bound cell wall proteins of Arabidopsis thaliana were identified using a proteomic and bioinformatic approach. An efficient protocol of extraction based on vacuum-infiltration of the tissues was developed. Several salts and a chelating agent were compared for their ability to extract cell wall proteins without releasing cytoplasmic contaminants. Of the 93 proteins that were identified, a large proportion (60%) was released by calcium chloride. From bioinformatics analysis, it may be predicted that most of them (87 out of 93) had a signal peptide, whereas only six originated from the cytoplasm. Among the putative apoplastic proteins, a high proportion (67 out of 87) had a basic pI. Numerous glycoside hydrolases and proteins with interacting domains were identified, in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and recognition phenomena. Ten proteinases were also found as well as six proteins with unknown functions. Comparison of the cell wall proteome of rosettes with the previously published cell wall proteome of cell suspension cultures showed a high level of cell specificity, especially for the different members of several large multigenic families.

Background: Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time.

The complete sequencing of the Arabidopsis thaliana genome allows the use of the recently developed mass spectrometry techniques to identify the cell wall proteins (CWPs). Most proteomic approaches depend on the quality of sample preparation. Extraction of CWPs is particularly complex since the proteins may be free in the apoplast or are embedded in a polysaccharide matrix where they are retained by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions, or cross-linked by covalent bonds. Specific and sequential extraction procedures thus need to be developed. We report on the sequential extraction of loosely bound CWPs from living A. thaliana cells in culture. Different salts and chelating agents were used for releasing the proteins from the wall. Their effects on the extraction of CWPs and on the integrity of the plasma membrane were evaluated. Bioinformatic software was used to identify proteins and to predict their sub-cellular localization. The obtained data show that the plasma membrane of cells in culture was easily damaged by some steps of the extraction procedure, leading to the release of increasing amounts of intracellular proteins. Nevertheless, we identified fifty CWPs among which thirteen were new proteins for the cell wall. In addition, 76% of these CWPs were basic proteins not resolved in two-dimensional (2-D) gel electrophoresis. The existence of two hypothetical proteins was confirmed. The structure of three proteins could be confirmed using mass spectrometry data.

Summary• Effects of two algal polysaccharides, laminarin and carrageenans, on defence responses and signalling in tobacco plants is presented. A possible role as defence elicitors is important in the context of the use of algal extracts as plant protectants.• The effect of the extracts was assessed after infiltration of tobacco leaves, and compared to the effect of a known elicitor of Phytophthora parasitica var. nicotianae ( Ppn ).• Of the two algal polysaccharides, only carrageenans efficiently induced signalling and defence gene expression in tobacco leaves, as observed with Ppn elicitor. λ -carrageenan, with its high sulphate content, proved the most active. Defence genes encoding sesquiterpene cylase, chitinase and proteinase inhibitor were induced locally, and the signalling pathways mediated by ethylene, jasmonic acid and salicylic acid, were triggered. Some effects lasted for at least a week.• λ -Carrageenan can elicit an array of plant defence responses, possibly through an effect of its high sulphate content. This helps clarify the mechanism of plant protection by algal extracts.

The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H 2 O 2 -independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90-kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm.The cDNA clones corresponding to the 90-kDa and 110-kDa proteins, lac90 and lac110, were obtained by a PCRbased approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with genespecific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.Keywords: laccase, poplar, lignification, dehydrogenation polymers.Lignins are complex aromatic heteropolymers that are derived from phenylpropanoid metabolism. They represent 30% of the Earth's woody biomass and are considered to have had a crucial role in the adaptation of land plants to the terrestrial habitat. However, many aspects concerning their biosynthesis and its regulation have yet to be elucidated. One striking illustration of this is that the enzyme(s) involved in the final polymerization of the p-hydroxycinnamyl alcohol precursors (monolignols) is still unknown. Freudenberg et al.[1] were first to demonstrate that a laccase (p-diphenol:oxygen oxidoreductase, EC 1.10.3.2) from extracts of the fungus, Agaricus campestris, and a laccase-like enzyme from cambial extracts of the gymnosperm, Araucaria excelsa, could catalyse coniferyl alcohol polymerization, thereby implicating laccases in lignification. However, when Nakamura [2] reported that laccase from Rhus vernicifera could not oxidize coniferyl alcohol and Harkin and Obst [3] showed that tree stems sections could not oxidize syringaldazine in the absence of exogenous H 2 O 2 , the proposed involvement of laccase in lignification was discounted. Peroxidase was thereafter considered to be exclusively involved in the dehydrogenative polymerization of lignin precursors.In recent years, convinc...

Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1–. In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1– exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors’ knowledge, this is the first functional characterization of CCR1 in a grass species.

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Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that is capable of growing in the presence of methanol as the sole carbon and energy source, but is also able to grow on a limited number of C(2), C(3), and C(4) compounds, for example succinate. This study provides a proteomic view of the cellular adaptation of M. extorquens AM1 to growth on methanol and succinate, respectively. Cytosolic proteins were separated by two-dimensional gel electrophoresis employing overlapping pH ranges and visualized by silver nitrate or fluorescence staining. A proteomic reference map containing 229 different proteins identified by peptide mass fingerprinting of tryptic fragments was established. Comparative proteome profiling of methanol- and succinate-grown cells led to the identification of 68 proteins that are induced under methylotrophic growth conditions in comparison to growth on succinate. This group includes most proteins known to be directly involved in methanol oxidation to CO(2) and in assimilation of one carbon units by the serine cycle as well as 18 proteins without any assigned function and two proteins with a predicted regulatory function. Furthermore, the proteome analysis revealed putative isoenzymes for formaldehyde-activating enzyme Fae, malyl-CoA lyase, malate-dehydrogenase, and fumarase, that need to be characterized functionally in future studies.