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The remarkable desiccation tolerance of the vegetative tissues in the resurrection species Craterostigma plantagineum (Hochst.) is favored by its unique cell wall folding mechanism that allows the ordered and reversible shrinking of the cells without damaging neither the cell wall nor the underlying plasma membrane. The ability to withstand extreme drought is also maintained in abscisic acid pre-treated calli, which can be cultured both on solid and in liquid culture media. Cell wall research has greatly advanced, thanks to the use of inhibitors affecting the biosynthesis of e.g., cellulose, since they allowed the identification of the compensatory mechanisms underlying habituation. Considering the innate cell wall plasticity of C. plantagineum , the goal of this investigation was to understand whether habituation to the cellulose biosynthesis inhibitors dichlobenil and isoxaben entailed or not identical mechanisms as known for non-resurrection species and to decipher the cell wall proteome of habituated cells. The results showed that exposure of C. plantagineum calli/cells triggered abnormal phenotypes, as reported in non-resurrection species. Additionally, the data demonstrated that it was possible to habituate Craterostigma cells to dichlobenil and isoxaben and that gene expression and protein abundance did not follow the same trend. Shotgun and gel-based proteomics revealed a common set of proteins induced upon habituation, but also identified candidates solely induced by habituation to one of the two inhibitors. Finally, it is hypothesized that alterations in auxin levels are responsible for the increased abundance of cell wall-related proteins upon habituation.

Cannabis sativa is an economically important crop providing bast fibres for the textile and biocomposite sector. Length is a fundamental characteristic determining the properties of bast fibres. Aquaporins, channel-forming proteins facilitating the passage of water, urea, as well as elements such as boron and silicon, are known to play a role in the control of fibre length in other species, like cotton. By mining the available genome, we here identify, for the first time, the aquaporin gene family of C. sativa. The analysis of published RNA-Seq data and targeted qPCR on a textile variety reveal an organ-specific expression of aquaporin genes. Computational analyses, including homology-based search, phylogeny and protein modelling, identify two NOD26-like intrinsic proteins harbouring the Gly-Ser-Gly-Arg (GSGR) aromatic/Arg selectivity filter and 108 amino acid NPA (Asn-Pro-Ala) spacing, features reported to be associated with silicon permeability. SIMS nano-analysis and silica extraction coupled to fluorescence microscopy performed on hemp plantlets reveal the presence of silicon in the bast fibres of the hypocotyl and in leaves. The accumulation of silica in the distal cell walls of bast fibres and in the basal cells of leaf trichomes is indicative of a mechanical role., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The assumption that cellulose degradation and assimilation can only be carried out by heterotrophic organisms was shattered in 2012 when it was discovered that the unicellular green alga, Chlamydomonas reinhardtii (Cr), can utilize cellulose for growth under CO₂-limiting conditions. Publications of genomes/transcriptomes of the colonial microalgae, Gonium pectorale (Gp) and Volvox carteri (Vc), between 2010⁻2016 prompted us to look for cellulase genes in these algae and to compare them to cellulases from bacteria, fungi, lower/higher plants, and invertebrate metazoans. Interestingly, algal catalytic domains (CDs), belonging to the family GH9, clustered separately and showed the highest (33⁻42%) and lowest (17⁻36%) sequence identity with respect to cellulases from invertebrate metazoans and bacteria, respectively, whereas the identity with cellulases from plants was only 27⁻33%. Based on comparative multiple alignments and homology models, the domain arrangement and active-site architecture of algal cellulases are described in detail. It was found that all algal cellulases are modular, consisting of putative novel cysteine-rich carbohydrate-binding modules (CBMs) and proline/serine-(PS) rich linkers. Two genes were found to encode a protein with a putative Ig-like domain and a cellulase with an unknown domain, respectively. A feature observed in one cellulase homolog from Gp and shared by a spinach cellulase is the existence of two CDs separated by linkers and with a C-terminal CBM. Dockerin and Fn-3-like domains, typically found in bacterial cellulases, are absent in algal enzymes. The targeted gene expression analysis shows that two Gp cellulases consisting, respectively, of a single and two CDs were upregulated upon filter paper addition to the medium.

The seed proteome of two traditional maize inbred lines (pb269 and pb369) contrasting in grain hardness and in preferable use for bread-making was evaluated. The pb269 seeds, of flint type (i.e., hard endosperm), are preferably used by manufacturers, while pb369 (dent, soft endosperm) is rejected. The hypothesis that the content and relative amounts of specific proteins in the maize flour are relevant for such discrimination of the inbred lines was tested. The flour proteins were sequentially extracted following the Osborne fractionation (selective solubilization), and the four Osborne fractions were submitted to two-dimensional electrophoresis (2DE). The total amount of protein extracted from the seeds was not significantly different, but pb369 flour exhibited significantly higher proportions of salt-extracted proteins (globulins) and ethanol-extracted proteins (alcohol-soluble prolamins). The proteome analysis allowed discrimination between the two inbred lines, with pb269 demonstrating higher heterogeneity than pb369. From the 967 spots (358 common to both lines, 208 specific to pb269, and 401 specific to pb369), 588 were submitted to mass spectrometry (MS). Through the combined use of trypsin and chymotrypsin it was possible to identify proteins in 436 spots. The functional categorization in combination with multivariate analysis highlighted the most discriminant biological processes (carbohydrate metabolic process, response to stress, chitin catabolic process, oxidation-reduction process) and molecular function (nutrient reservoir activity). The inbred lines exhibited quantitative and qualitative differences in these categories. Differences were also revealed in the amounts, proportions, and distribution of several groups of storage proteins, which can have an impact on the organization of the protein body and endosperm hardness. For some proteins (granule-bound starch synthase-1, cyclophilin, zeamatin), a change in the protein solubility rather than in the total amount extracted was observed, which reveals distinct in vivo associations and/or changes in binding strength between the inbred lines. Our approach produced information that relates protein content, relative protein content, and specific protein types to endosperm hardness and to the preferable use for "broa" bread-making.

The occurrence of high-ozone concentrations during drought episodes is common considering that they are partially caused by the same meteorological phenomena. It was suggested that mild drought could protect plants against ozone-induced damage by causing the closure of stomata and preventing the entry of ozone into the leaves. The present experiment attempts to create an overview of the changes in cellular processes in response to ozone, mild drought and a combined treatment based on the use of 2D-DiGE to compare the involved proteins, and a number of supporting analyses. Morphological symptoms were worst in the combined treatment, indicating a severe stress, but fewer proteins were differentially abundant in the combined treatment than for ozone alone. Stomatal conductance was slightly lowered in the combined treatment. Shifts in carbon metabolism indicated that the metabolism changed to accommodate for protective measures and changes in the abundance of proteins involved in redox protection indicated the presence of an oxidative stress. This study allowed identifying a set of proteins that changed similarly during ozone and drought stress, indicative of crosstalk in the molecular response of plants exposed to these stresses. The abundance of other key proteins changed only when the plants are exposed to specific conditions. Together this indicates the coexistence of generalized and specialized responses to different conditions., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ability of plants to recover from freeze-thaw injury is a critical component of freeze-thaw stress tolerance. To investigate the molecular basis of freeze-thaw recovery, here we compared the proteomes of onion scales from unfrozen control (UFC), freeze-thaw injured (INJ), and post-thaw recovered (REC) treatments. Injury-related proteins (IRPs) and recovery-related proteins (RRPs) were differentiated according to their accumulation patterns. Many IRPs decreased right after thaw without any significant re-accumulation during post-thaw recovery, while others were exclusively induced in INJ tissues. Most IRPs are antioxidants, stress proteins, molecular chaperones, those induced by physical injury or proteins involved in energy metabolism. Taken together, these observations suggest that while freeze-thaw compromises the constitutive stress protection and energy supply in onion scales, it might also recruit 'first-responders' (IRPs that were induced) to mitigate such injury. RRPs, on the other hand, are involved in the injury-repair program during post-thaw environment conducive for recovery. Some RRPs were restored in REC tissues after their first reduction right after thaw, while others exhibit higher abundance than their 'constitutive' levels. RRPs might facilitate new cellular homeostasis, potentially by re-establishing ion homeostasis and proteostasis, cell-wall remodelling, reactive oxygen species (ROS) scavenging, defence against possible post-thaw infection, and regulating the energy budget to sustain these processes., (© 2012 Blackwell Publishing Ltd.)

Young poplar plants were exposed to different heat regimes, a rapid heat constraint at 42°C (heat shock HS) alone or preceded by a stepwise increase in temperature (heat gradient HG). Proteomics analyses were carried out on both leaf and cambial tissues. The responses of both tissues were compared and linked to morphological and physiological observations. Both heat treatments negatively affected the photosynthetic rate while increasing the stomatal conductance. In the leaf, the HS impacted some photosynthetic proteins, and particularly induced an increase in abundance of proteins of the oxygen evolving complexes. On the other hand, the HG reduced carbohydrate metabolism and induced mainly an increase in germin-like proteins. In the cambial zone, the HS caused a decrease in sucrose synthase content and in enzymes related to protein synthesis. The main effect of HG was the accumulation of thaumatin-like proteins as well as an increase in the abundance of proteins involved in carbohydrate metabolism. Further, both tissues underwent changes in the content of heat shock proteins, but more importantly, of peroxiredoxins. The results show more sustainable changes in leaf and cambial proteomes in response to HS compared to HG., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Cork (phellem) formation in Quercus suber stem was studied by proteomic analysis of young shoots of increasing age (Y0, Y1 and Y4) and recently-formed phellem (Y8Ph) and xylem (Y8X) from an 8-year-old branch. In this study 99 proteins were identified, 45 excised from Y8X and 54 from Y8Ph. These ones, specifically associated with phellem, are of "carbohydrate metabolism" (28%), "defence" (22%), "protein folding, stability and degradation" (19%), "regulation/signalling" (11%), "secondary metabolism" (9%), "energy metabolism" (6%), and "membrane transport" (2%). The identification in phellem of galactosidases, xylosidases, apiose/xylose synthase, laccases and diphenol oxidases suggests intense cell wall reorganization, possibly with participation of hemicellulose/pectin biosynthesis and phenol oxidation. The identification of proteasome subunits, heat shock proteins, cyclophylin, subtilisin-like proteases, 14-3-3 proteins, Rab2 protein and enzymes interacting with nucleosides/nucleic acids gives additional evidence for cellular reorganization, involving cellular secretion, protein turnover regulation and active control processes. The high involvement in phellem of defence proteins (thioredoxin-dependent peroxidase, glutathione-S-transferase, SGT1 protein, cystatin, and chitinases) suggests a strong need for cell protection from the intense stressful events occurring in active phellem, namely, desiccation, pests/disease protection, detoxification and cell death. Identically, highly enhanced defence functions were previously reported for potato periderm formation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties. Via Principal Component Analysis (PCA) and hierarchical clustering we were able to blindly classify the different varieties according to their presumed genome constitution. We report for the first time the application of an automated approach for the derivatization of peptides for facilitated MS/MS de novo sequence determination. We conclude that the proteome does not always correspond to the presumed genome formulae and that proteomics is a powerful tool to characterize varieties. The observations at the protein level provide good indications for a more complex genome structure and genomic rearrangement in some banana varieties., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

Cardosins are model plant aspartic proteases, a group of proteases that are involved in cell death events associated with plant senescence and stress responses. They are synthesized as single-chain zymogens, and subsequent conversion into two-chain mature enzymes is a crucial step in the regulation of their activity. Here we describe the activation and proteolytic processing of recombinant procardosin A. The cleavage sites involved in this multi-step autocatalytic process were determined, some of them using a novel method for C-terminal sequence analysis. Even though the two-chain recombinant enzyme displayed similar properties as natural cardosin A, a single-chain mutant form was engineered based on the processing results and produced in Escherichia coli. Determination of its primary specificity using two combinatorial peptide libraries revealed that this mutant form behaved like the natural enzyme. The primary specificity of the enzyme closely resembles those of cathepsin D and plasmepsins, suggesting that cardosin A shares the same peptide scissile bond preferences of its vacuolar/lysosomal mammalian and protozoan homologues.

Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in pep tide fractionation amenable to MS analysis are as well discussed. [ABSTRACT FROM AUTHOR]