Comprehensive analysis of the membrane phosphoproteome regulated by oligogalacturonides in Arabidopsis thaliana

Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity

Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea

Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced in response to pathogen invasion attempts at the infection site and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). In this protocol the detection and quantification of camalexin produced in Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea is described

Changes in the microsomal proteome of tomato fruit during ripening

The variations in the membrane proteome of tomato fruit pericarp during ripening have been investigated by mass spectrometry-based label-free proteomics. Mature green (MG30) and red ripe (R45) stages were chosen because they are pivotal in the ripening process: MG30 corresponds to the end of cellular expansion, when fruit growth has stopped and fruit starts ripening, whereas R45 corresponds to the mature fruit. Protein patterns were markedly different: among the 1315 proteins identified with at least two unique peptides, 145 significantly varied in abundance in the process of fruit ripening. The subcellular and biochemical fractionation resulted in GO term enrichment for organelle proteins in our dataset, and allowed the detection of low-abundance proteins that were not detected in previous proteomic studies on tomato fruits. Functional annotation showed that the largest proportion of identified proteins were involved in cell wall metabolism, vesicle-mediated transport, hormone biosynthesis, secondary metabolism, lipid metabolism, protein synthesis and degradation, carbohydrate metabolic processes, signalling and response to stress

Dampening the DAMPs: how plants maintain the homeostasis of cell wall molecular patterns and avoid hyper-immunity

Several oligosaccharide fragments derived from plant cell walls activate plant immunity and behave as typical damage-associated molecular patterns (DAMPs). Some of them also behave as negative regulators of growth and development, and due to their antithetic effect on immunity and growth, their concentrations, activity, time of formation, and localization is critical for the so-called “growth-defense trade-off.” Moreover, like in animals, over accumulation of DAMPs in plants provokes deleterious physiological effects and may cause hyper-immunity if the cellular mechanisms controlling their homeostasis fail. Recently, a mechanism has been discovered that controls the activity of two well-known plant DAMPs, oligogalacturonides (OGs), released upon hydrolysis of homogalacturonan (HG), and cellodextrins (CDs), products of cellulose breakdown. The potential homeostatic mechanism involves specific oxidases belonging to the family of berberine bridge enzyme-like (BBE-like) proteins. Oxidation of OGs and CDs not only inactivates their DAMP activity, but also makes them a significantly less desirable food source for microbial pathogens. The evidence that oxidation and inactivation of OGs and CDs may be a general strategy of plants for controlling the homeostasis of DAMPs is discussed. The possibility exists of discovering additional oxidative and/or inactivating enzymes targeting other DAMP molecules both in the plant and in animal kingdoms

Working with Tweets: The Effectiveness of Lean Communication in Collaborative Problem-Solving

Among social network sites and tools, Twitter and other microblogging tools are characterized by their particularly "lean" communication form, based on quick and short messages, particularly appealing for personal use by the so called "generation Y". In this paper we investigate whether lean communication - and particularly Twitter - can be used as a tool for teamwork in organizations. Therefore, our research question is focused on whether Twitter could represent a viable communication tool in the future enterprise for collaborative problem solving. More specifically, whether the 140 characters limit would actually make a difference. To answer this research question, we propose here a comparative study of Twitter, a well-known SNS for microblogging, and Skype, a typical communication platform for individuals and organizations. The laboratory experiment method was selected in order to conduct a rigorous comparison with well- defined measurements. Different teams of the same size carry out a well structured problem- solving task communicating via Twitter or Skype. Comparative group performance is measured in terms of task completion time. Our results show that the lower volume of information exchanged via Twitter does not negatively affect group performance: Twitter teams are just as effective as Skype teams. These results can be partly explained in terms of media richness theory, but more interestingly they may be related to the different group dynamics within teams using different systems, and to further dimensions of investigation including the subjects, the task, the technological features and the organizational coordination modes, disclosing new paths for further research

Isolation and characterization of oxidizedoligogalacturonides: meccanism of dampening of damps

Oligogalacturonides (OGs) released upon partial degradation of homogalacturonan, are a well-known class of Damage-Associated Molecular Patterns (DAMPs). Besides inducing immunity, OGs negatively affect plant growth by antagonizing auxin responses. Because the recognition of DAMPs poses the intrinsic risk of activating an exaggerated response that may impair plant survival, dampening mechanisms of DAMPs should exist. Transgenic Arabidopsis plants (OGM plants) expressing a chimeric protein called "OGmachine" accumulate oligogalacturonides (OGs) in their tissues and exhibit enhanced resistance to a variety of pathogens; however the growth of these plants is severely impaired. The prolonged release of OGs triggers defense responses that in the long term are deleterious for the plant. We used the OGM plants as a tool to investigate a possible regulatory mechanism by searching for elicitor-inactive OGs that may derive from elicitor-active OGs through an enzymatic modification. By analyzing the OGs produced in the transgenic plants, modified OGs were isolated. The nature of the modification was investigated by electrospray ionization mass spectrometry and resulted to be the oxidation to galactaric acid of the residue at the reducing end of OGs (oxOGs). OxOGs were tested for their ability to induce defense responses and antagonize auxin responses. In all experiments, they were inactive as compared to the corresponding typical OGs. We succeeded to isolate and characterize one of the enzymes that causes the inactivation of OGs: it is a FAD binding oxidase, that we named OGOX1, capable of producing elicitor-inactive oxidized OGs and H2O2

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects

Degradazione della pectina nella parete vegetale: implicazioni fisiopatologiche e applicazioni biotecnologiche

La parete cellulare è una complessa struttura delle cellule vegetali che, oltre a determinarne la forma e rigidità, costituisce un importante elemento di regolazione della crescita e del differenziamento delle piante. Rappresenta inoltre l’interfaccia tra la cellula e l’ambiente e funziona come una barriera fisica di protezione dagli stress abiotici e biotici. La parete è un deposito di segnali regolatori che vengono rilasciati durante una infezione o in seguito al danno meccanico. La struttura delle pareti cellulari è sottoposta a un costante rimodellamento in quanto deve adattarsi alle esigenze di volta in volta prevalenti. La pectina in particolare influenza la rigidità della parete e l’adesione cellula-cellula, ed è il primo componente che viene modificato durante l’attacco dei patogeni o durante lo sviluppo. Pertanto, le piante hanno sviluppato un sistema sofisticato, principalmente incentrato sulla pectina, per monitorare la composizione della parete cellulare e regolarne le modifiche. (PIMS:"sistema di monitoraggio dell’integrità della pectina"). Scopo della mia ricerca è stato quello comprendere come le modifiche della pectina possano giocare un ruolo nel mantenere un corretto equilibrio tra difesa e sviluppo, con particolare attenzione all’impatto di queste modifiche sull’utilizzo delle biomasse vegetali per applicazioni industriali come la produzione di biocarburanti. Nella prima parte del lavoro mi sono occupata di valutare se l’espressione controllata di enzimi pectici in planta potesse contribuire a migliorare la resistenza ai patogeni e la saccarificazione della biomassa vegetale senza interferire nel normale sviluppo della pianta. Nella seconda parte del mio lavoro l’obiettivo è stato di approfondire la comprensione della funzione degli oligogalatturonidi (OG), una classe importante di elicitori rilasciati dalla pectina della parete cellulare vegetale per azione di poligalatturonasi microbiche durante l’infezione. Abbiamo generato delle piante transgeniche che esprimono, sotto il controllo di un promotore inducibile, una proteina chimerica capace di produrre OG, chiamata per l’appunto OG machine (OGM) , per studiare come i livelli di OG nel tessuto regolano, a seconda della concentrazione, il compromesso tra crescita e difesa. Nella terza parte della tesi, l’analisi proteomica e fosfoproteomica delle membrane di Arabidopsis thaliana in seguito al trattamento con OG esogeni ha permesso di studiare le risposte precoci della pianta e dissezionare la via di segnalazione attivata da questi elicitori.The cell wall is a complex structure of plant cells which determines their shape and rigidity, and is an important element in the regulation of plant growth and differentiation. It also represents the interface between the cell and its environment and acts as a physical barrier to protect it against abiotic and biotic stresses. The wall is a deposit of regulatory signals that are released during infections or due to mechanical damage. The structure of the cell walls is in a constant process of remodeling as it adapts to the prevailing functional requirements. Pectin in particular influences cell wall rigidity as well as cell–cell adhesion , and is the first component that is modified during the attack of pathogens or during development. Therefore, plants have developed a sophisticated system mainly focused on pectin, to monitor the composition of the cell wall and regulate its modifications. ( PIMS " system for monitoring the integrity of the pectin "). The aim of my research was to understand how pectin modification may play a role in maintaining a proper balance between defense and development, with particular attention to the impact of these modifications on the use of plant biomass for industrial applications such as biofuel production . In the first part of the work I assessed whether the controlled expression of pectic enzymes in planta could help to improve the resistance to pathogens and the saccharification of plant biomass without interfering with the normal development of the plant . In the second part of my work , the objective was to deepen our understanding of the function of oligogalacturonides (OG), an important class of elicitors released from the cell wall pectin by the action of polygalacturonases during microbial infection. We generated transgenic plants that express, under the control of an inducible promoter, a chimeric protein capable of producing OG, called OG machine ( OGM), to study how the levels of OGs in the tissue regulate, depending on the concentration, the growth/defense trade off. In the third part of the thesis, proteomic and phosphoproteomic analysis of the membranes of Arabidopsis thaliana in response to treatment with exogenous OG has allowed us to study the early response of the plant and dissect the signaling pathway activated by these elicitors