Unraveling the Mechanisms of Efficient Phosphorus Utilization in Popcorn (Zea mays L. var. everta): Insights from Proteomic and Metabolite Analysis.

The expansion of agriculture and the need for sustainable practices drives breeders to develop plant varieties better adapted to abiotic stress such as nutrient deficiency, which negatively impacts yields. Phosphorus (P) is crucial for photosynthesis and plant growth, but its availability in the soil is often limited, hampering crop development. In this study, we examined the response of two popcorn inbred lines, L80 and P7, which have been characterized previously as P-use inefficient and P-use efficient, respectively, under low (stress) and high P (control) availability. Physiological measurements, proteomic analysis, and metabolite assays were performed to unravel the physiological and molecular responses associated with the efficient use of P in popcorn. We observed significant differences in protein abundances in response to the P supply between the two inbred lines. A total of 421 differentially expressed proteins (DEPs) were observed in L80 and 436 DEPs in P7. These proteins were involved in photosynthesis, protein biosynthesis, biosynthesis of secondary metabolites, and energy metabolism. In addition, flavonoids accumulated in higher abundance in P7. Our results help us understand the major components of P utilization in popcorn, providing new insights for popcorn molecular breeding programs.

Preliminary Insights of Brazilian Snake Venom Metalloproteomics.

Snakebite envenoming is one of the most significantly neglected tropical diseases in the world. The lack of diagnosis/prognosis methods for snakebite is one of our motivations to develop innovative technological solutions for Brazilian health. The objective of this work was to evaluate the protein and metallic ion composition of Crotalus durissus terrificus, Bothrops jararaca, B. alternatus, B. jararacussu, B. moojeni, B. pauloensis, and Lachesis muta muta snake venoms. Brazilian snake venoms were subjected to the shotgun proteomic approach using mass spectrometry, and metal ion analysis was performed by atomic spectrometry. Shotgun proteomics has shown three abundant toxin classes (PLA2, serine proteases, and metalloproteinases) in all snake venoms, and metallic ions analysis has evidenced that the Cu2+ ion is present exclusively in the L. m. muta venom; Ca2+ and Mg2+ ions have shown a statistical difference between the species of Bothrops and Crotalus genus, whereas the Zn2+ ion presented a statistical difference among all species studied in this work. In addition, Mg2+ ions have shown 42 times more in the C. d. terrificus venom when compared to the average concentration in the other genera. Though metal ions are a minor fraction of snake venoms, several venom toxins depend on them. We believe that these non-protein fractions are capable of assisting in the development of unprecedented diagnostic devices for Brazilian snakebites.

Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress.

Bacillus toyonensis SFC 500-1E is a member of the consortium SFC 500-1 able to remove Cr(VI) and simultaneously tolerate high phenol concentrations. In order to elucidate mechanisms utilized by this strain during the bioremediation process, the differential expression pattern of proteins was analyzed when it grew with or without Cr(VI) (10 mg/L) and Cr(VI) + phenol (10 and 300 mg/L), through two complementary proteomic approaches: gel-based (Gel-LC) and gel-free (shotgun) nanoUHPLC-ESI-MS/MS. A total of 400 differentially expressed proteins were identified, out of which 152 proteins were down-regulated under Cr(VI) and 205 up-regulated in the presence of Cr(VI) + phenol, suggesting the extra effort made by the strain to adapt itself and keep growing when phenol was also added. The major metabolic pathways affected include carbohydrate and energetic metabolism, followed by lipid and amino acid metabolism. Particularly interesting were also ABC transporters and the iron-siderophore transporter as well as transcriptional regulators that can bind metals. Stress-associated global response involving the expression of thioredoxins, SOS response, and chaperones appears to be crucial for the survival of this strain under treatment with both contaminants. This research not only provided a deeper understanding of B. toyonensis SFC 500-1E metabolic role in Cr(VI) and phenol bioremediation process but also allowed us to complete an overview of the consortium SFC 500-1 behavior. This may contribute to an improvement in its use as a bioremediation strategy and also provides a baseline for further research.

Protein Succinylation and Malonylation as Potential Biomarkers in Schizophrenia.

Two protein post-translational modifications, lysine succinylation and malonylation, are implicated in protein regulation, glycolysis, and energy metabolism. The precursors of these modifications, succinyl-CoA and malonyl-CoA, are key players in central metabolic processes. Both modification profiles have been proven to be responsive to metabolic stimuli, such as hypoxia. As mitochondrial dysfunction and metabolic dysregulation are implicated in schizophrenia and other psychiatric illnesses, these modification profiles have the potential to reveal yet another layer of protein regulation and can furthermore represent targets for biomarkers that are indicative of disease as well as its progression and treatment. In this work, data from shotgun mass spectrometry-based quantitative proteomics were compiled and analyzed to probe the succinylome and malonylome of postmortem brain tissue from patients with schizophrenia against controls and the human oligodendrocyte precursor cell line MO3.13 with the dizocilpine chemical model for schizophrenia, three antipsychotics, and co-treatments. Several changes in the succinylome and malonylome were seen in these comparisons, revealing these modifications to be a largely under-studied yet important form of protein regulation with broad potential applications.

Proteomics and Schizophrenia: The Evolution of a Great Partnership.

The mass spectrometer is an instrument that observes particular masses of molecules of interest. Over the past century, it has grown to become a highly sensitive and robust tool in laboratorial and clinical research to identify and quantify thousands of proteins in a given sample in an unbiased manner leading to the quick rise in its use. This unbiased and high-throughput nature is extremely important in discovery-based studies, since no preset targets can be selected, as is the case with several other proteomic methods. In studying multifactorial diseases such as schizophrenia, mass-spectrometry-based proteomics has been frequently used and new improvements to the technique have been quickly taken advantage of. Over the past 15 years, mass spectrometry has evolved greatly, and with it, the proteomic analyses and data have evolved. In this chapter, a brief history of the evolution of mass spectrometry is covered along with how schizophrenia research has grown alongside this valuable methodology.

Assessing proteolytic events in bioinformatic reanalysis of public secretome data from melanoma cell lines.

Autocrine and paracrine signals are of paramount importance in both normal and oncogenic events and the composition of such secreted molecular signals (i.e the secretome) designate the communication status of cells. In this context, the analysis of post-translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as proteolytic processing. In the present study, we have performed a bioinformatic reanalysis of public proteomics data on melanoma cell line secretomes, changing database searching parameters to allow for the identification of proteolytic events generated by active proteases. Such approach enabled the identification of proteolytic signatures which suggested active proteases and whose expression profiles might be targeted in patient tissues or liquid biopsies, as well as their cleaved substrates. Although N-terminomics approaches continue to be the method of choice for the evaluation of proteolytic signaling events in complex samples, the simple approach performed in this work resulted in the gain of biological insights derived from shotgun proteomics data.

ExVe: The knowledge base of orthologous proteins identified in fungal extracellular vesicles.

Extracellular vesicles (EVs) are double-membrane particles associated with intercellular communication. Since the discovery of EV production in the fungus Cryptococcus neoformans, the importance of EV release in its physiology and pathogenicity has been investigated. To date, few studies have investigated the proteomic content of EVs from multiple fungal species. Our main objective was to use an orthology approach to compare proteins identified by EV shotgun proteomics in 8 pathogenic and 1 nonpathogenic species. Using protein information from the UniProt and FungiDB databases, we integrated data for 11,433 hits in fungal EVs with an orthology perspective, resulting in 3,834 different orthologous groups. OG6_100083 (Hsp70 Pfam domain) was the unique orthologous group that was identified for all fungal species. Proteins with this protein domain are associated with the stress response, survival and morphological changes in different fungal species. Although no pathogenic orthologous group was found, we identified 5 orthologous groups exclusive to S. cerevisiae. Using the criteria of at least 7 pathogenic fungi to define a cluster, we detected the 4 unique pathogenic orthologous groups. Taken together, our data suggest that Hsp70-related proteins might play a key role in fungal EVs, regardless of the pathogenic status. Using an orthology approach, we identified at least 4 protein domains that could be novel therapeutic targets against pathogenic fungi. Our results were compiled in the herein described ExVe database, which is publicly available at http://exve.icc.fiocruz.br.

Design and evaluation of synthetic bacterial consortia for optimized phenanthrene degradation through the integration of genomics and shotgun proteomics.

Two synthetic bacterial consortia (SC) composed of bacterial strains Sphingobium sp. (AM), Klebsiella aerogenes (B), Pseudomonas sp. (Bc-h and T), Burkholderia sp. (Bk) and Inquilinus limosus (Inq) isolated from a natural phenanthrene (PHN)-degrading consortium (CON) were developed and evaluated as an alternative approach to PHN biodegradation in bioremediation processes. A metabolic network showing the potential role of strains was reconstructed by in silico study of the six genomes and classification of dioxygenase enzymes using RHObase and AromaDeg databases. Network analysis suggested that AM and Bk were responsible for PHN initial attack, while Inq, B, T and Bc-h would degrade PHN metabolites. The predicted roles were further confirmed by physiological, RT-qPCR and metaproteomic assays. SC-1 with AM as the sole PHN degrader was the most efficient. The ecological roles inferred in this study can be applied to optimize the design of bacterial consortia and tackle the biodegradation of complex environmental pollutants.

Proteomic changes in Trypanosoma cruzi epimastigotes treated with the proapoptotic compound PAC-1.

Apoptosis is a highly regulated process of cell death in metazoans. Therefore, understanding the biochemical changes associated with apoptosis-like death in Trypanosoma cruzi is key to drug development. PAC-1 was recently shown to induce apoptosis in T. cruzi; with this as motivation, we used quantitative proteomics to unveil alterations of PAC-1-treated versus untreated epimastigotes. The PAC-1 treatment reduced the abundance of putative vesicle-associated membrane protein, putative eukaryotic translation initiation factor 1 eIF1, coatomer subunit beta, putative amastin, and a putative cytoskeleton-associated protein. Apoptosis-like signaling also increases the abundance of proteins associated with actin cytoskeleton remodeling, cell polarization, apoptotic signaling, phosphorylation, methylation, ergosterol biosynthesis, vacuolar proteins associated with autophagy, and flagellum motility. We shortlist seventeen protein targets for possible use in chemotherapy for Chagas disease. Almost all differentially abundant proteins belong to a family of proteins previously associated with apoptosis in metazoans, suggesting that the apoptotic pathway's key functions have been preserved from trypanosomatids and metazoans. SIGNIFICANCE: Approximately 8 million people worldwide are infected with Trypanosoma cruzi. The treatment of Chagas disease comprises drugs with severe side effects, thus limiting their application. Thus, developing new pharmaceutical solutions is relevant, and several molecules targeting apoptosis are therapeutically efficient for parasitic, cardiac, and neurological diseases. Apoptotic processes lead to specific morphological features that have been previously observed in T. cruzi. Here, we investigate changes in epimastigotes' proteomic profile treated with the proapoptotic compound PAC-1, providing data concerning the regulation of both metabolic and cellular processes in nonmetazoan apoptotic cells. We shortlist seventeen protein target candidates for use in chemotherapy for Chagas disease.

Using a toxicoproteomic approach to investigate the effects of thiamethoxam into the brain of Apis mellifera.

Neonicotinoids have been described as toxic to bees. In this context, the A. mellifera foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100: 0,0227 ng de thiamethoxam/µL-1 diet), a neurotoxic insecticide, for 8 days; and it was decided to investigate the insecticide effect on the brain by a shotgun proteomic approach followed by label-free quantitative-based proteomics. A total of 401 proteins were identified in the control group (CG); and a total of 350 proteins in the thiamethoxam exposed group (TMX). Quantitative proteomics data showed up 251 proteins with significant quantitative values in the TMX group. These findings demonstrated the occurrence of shared and unique proteins with altered expression in the TMX group, such as ATP synthase subunit beta, heat shock protein cognate 4, spectrin beta chain-like, mushroom body large-type Kenyon cell-specific protein 1-like, tubulin alpha-1 chain-like, arginine kinase, epidermal growth factor receptor, odorant receptor, glutamine synthetase, glutamate receptor, and cytochrome P450 4c3. Meanwhile, the proteins that were expressed uniquely in the TMX group are involved mainly in the phosphorylation, cellular protein modification, and cell surface receptor signalling processes. Interaction network results showed that identified proteins are present in five different metabolic pathways - oxidative stress, cytoskeleton control, visual process, olfactory memory, and glutamate metabolism. Our scientific outcomes demonstrated that a sublethal concentration of thiamethoxam can impair biological processes and important metabolic pathways, causing damage to the nervous system of bees, and in the long term, can compromise the nutrition and physiology of individuals from the colony.

Quantitative proteomic dataset from oro- and naso-pharyngeal swabs used for COVID-19 diagnosis: Detection of viral proteins and host's biological processes altered by the infection.

Since January 2020, the world is facing the COVID-19 pandemic caused by SARS-CoV-2. In a big effort to cope with this outbreak, two Uruguayan institutions, Institut Pasteur de Montevideo and Universidad de la República, have developed and implemented a diagnosis pipeline based on qRT-PCR using entirely local resources. In this context, we performed comparative quantitative proteomic analysis from oro- and naso-pharyngeal swabs used for diagnosis. Tryptic peptides obtained from five positive and five negative samples were analysed by nano-LC-MS/MS using a Q-Exactive Plus mass spectrometer. Data analysis was performed using PatternLab for Proteomics software. From all SARS-CoV-2 positive swabs we were able to detect peptides of the SARS-CoV-2 nucleoprotein that encapsulates and protect the RNA genome. Additionally, we detected an average of 1100 human proteins from each sample. The most abundant proteins exclusively detected in positive swabs were "Guanylate-binding protein 1", "Tapasin" and "HLA class II histocompatibility antigen DR beta chain". The biological processes overrepresented in infected host cells were "SRP-dependent cotranslational protein targeting to membrane", "nuclear-transcribed mRNA catabolic process, nonsense-mediated decay", "viral transcription" and "translational initiation". Data is available via ProteomeXchange with identifier PXD020394. We expect that this data can contribute to the future development of mass spectrometry based approaches for COVID-19 diagnosis. Also, we share this preliminary proteomic characterization concerning the host response to infection for its reuse in basic investigation.

Shotgun proteomics of Strongyloides venezuelensis infective third stage larvae: Insights into host-parasite interaction and novel targets for diagnostics.

Strongyloides venezuelensis is an important alternative source of antigen for the serologic diagnosis of human strongyloidiasis. Proteomics techniques applied to the analysis of the protein content of infective third stage larvae (iL3) of S. venezuelensis provide a powerful tool for the discovery of new candidates for immunodiagnosis. This study presents an overview of the protein iL3 S. venezuelensis focusing on the diagnosis of strongyloidiasis. A total of 877 proteins were identified by shotgun proteomics. Many of these proteins are involved in different cellular processes, metabolic as well as structural maintenance. Our results point to a catalog of possible diagnostic targets for human strongyloidiasis and highlight the need for evaluation of uncharacterized proteins, especially the proteins within the CAP domain, transthyretin, and BTPI inhibitor domains, as a repertoire as yet unexplored in the context of strongyloidiasis diagnostic markers. We believe that the protein profile presented in this shotgun analysis extends our understanding of the protein composition within the Strongyloides genus, opening up new perspectives for research on biomarkers that may help with the diagnosis of human strongyloidiasis. Data are available via ProteomeXchange with identifier PXD013703.

Shotgun proteomic analysis of Bordetella parapertussis provides insights into the physiological response to iron starvation and potential new virulence determinants absent in Bordetella pertussis.

Bordetella parapertussis is one of the pathogens that cause whooping cough. Even though its incidence has been rising in the last decades, this species remained poorly investigated. This study reports the first extensive proteome analysis of this bacterium. In an attempt to gain some insight into the infective phenotype, we evaluated the response of B. parapertussis to iron starvation, a critical stress the bacteria face during infection. Among other relevant findings, we observed that the adaptation to this condition involves significant changes in the abundance of two important virulence factors of this pathogen, namely, adenylate cyclase and the O-antigen. We further used the proteomic data to search for B. parapertussis proteins that are absent or classified as pseudogenes in the genome of Bordetella pertussis to unravel differences between both whooping cough causative agents. Among them, we identified proteins involved in stress resistance and virulence determinants that might help to explain the differences in the pathogenesis of these species and the lack of cross-protection of current acellular vaccines. Altogether, these results contribute to a better understanding of B. parapertussis biology and pathogenesis. SIGNIFICANCE: Whooping cough is a reemerging disease caused by both Bordetella pertussis and Bordetella parapertussis. Current vaccines fail to induce protection against B parapertussis and the incidence of this species has been rising over the years. The proteomic analysis of this study provided relevant insights into potential virulence determinants of this poorly-studied pathogen. It further identified proteins produced by B. parapertussis not present in B. pertussis, which might help to explain both the differences on their respective infectious process and the current vaccine failure. Altogether, the results of this study contribute to the better understanding of B. parapertussis pathogenesis and the eventual design of improved preventive strategies against whooping cough.

Label-Free Quantitative Proteomics of Enriched Nuclei from Sugarcane (Saccharum ssp) Stems in Response to Drought Stress.

Drought is considered the major abiotic stress limiting crop productivity. This study seeks to identify proteins involved in the drought response in sugarcane stems submitted to drought stress. The integration of nuclei enrichment sample preparation with the shotgun proteomic approach results in great coverage of the sugarcane stem proteome with 5381 protein groups identified. A total of 1204 differentially accumulated proteins are detected in response to drought, among which 586 and 618 are increased and reduced in abundance, respectively. A total of 115 exclusive proteins are detected, being 41 exclusives of drought-stressed plants and 74 exclusives of control plants. In the control plants, most of these proteins are related to cell wall metabolism, indicating that drought affects negatively the cell wall metabolism. Also, 37 transcription factors (TFs) are identified, which are low abundant nuclear proteins and are differentially accumulated in response to drought stress. These TFs are associated to protein domains such as leucine-rich (bZIP), C2H2, NAC, C3H, LIM, Myb-related, heat shock factor (HSF) and auxin response factor (ARF). Increased abundance of chromatin remodeling and RNA processing proteins are also observed. It is suggested that these variations result from an imbalance of protein synthesis and degradation processes induced by drought.

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics.

Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence.

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra-pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc - Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max-exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT-PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.

Manganese alters expression of proteins involved in the oxidative stress of Meyerozyma guilliermondii.

Organisms, in general, respond to environmental stress by altering their pattern of protein expression (proteome), as an alternative to growing in stressful conditions. A strain of Meyerozyma guilliermondii resistant to manganese was isolated from a sample of water collected from mine drainage in southeastern Minas Gerais (Brazil), and demonstrated manganese detoxification capacity. Protein extracts containing the soluble fractions were obtained after growth of the strain in the absence and presence of MnSO4. Tryptic peptides recovered from samples were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Shotgun/bottom-up analyses of the soluble fractions revealed a total of 1257 identified molecules. Treatment with Mn did not affect the growth of yeast but induced changes in the protein profile, with 117 proteins expressed in the absence of Mn and 69 expressed only in its presence. Most of these are annotated as related to DNA repair, oxidoreductase activity, and remodeling of gene expression. This is the first proteomic report of M. guilliermondii, with promising characteristics for Mn bioremediation, and the first of the genus Meyerozyma. This proteomic characterization may help in the understanding of molecular regulatory mechanisms associated with tolerance to excess Mn, and the potential use of biomass in bioremediation processes. SIGNIFICANCE: Environmental pollution by heavy metals such as manganese (Mn2+) has increased as it is a by-product of the mining industry and a potential environmental contaminant. Many studies have explored the use of bacteria for manganese bioremediation, but yeasts have emerged as a promising alternative, displaying faster growth and greater removal efficiency. Previous works of our laboratory showed that Meyerozyma guilliermondii, a non-pathogenic haploid yeast (ascomycete), has excellent removal and accumulation capacity of Mn2+, potentially useful in bioremediation. Nowadays efforts have been devoted to understanding the physiology of metal hyperaccumulation to gain insights into the molecular basis of hyperaccumulation. To obtain a comprehensive understanding of the molecular mechanism of Mn2+ hyperaccumulation in M. guilliermondii, proteomic approaches were employed yielding the first compositional proteomic map of total soluble proteins and their differential expression in the presence of Mn2+. We believe our findings are of biotechnological interest concerning the utilization of M. guilliermondii for bioremediation purposes.

Stress and cell cycle regulation during somatic embryogenesis plays a key role in oil palm callus development.

Oil palm is an oleaginous plant of relevant economic importance since its fruits are rich in vegetable oil. These plants have a single apical meristem and the main method for vegetative propagation is somatic embryogenesis. The aim of this study was to identify differentially abundant proteins from oil palm genotypes contrasting in the capacity of embryogenic competence acquisition, using shotgun proteomics. Oil palm leaves were subjected to callus induction and the material was collected in biological triplicates at 14 and 90 days of callus induction. LC-MS/MS analysis was performed and revealed a total of 4695 proteins. Responsive and non-responsive genotypes were compared at 14 and 90 days of callus induction and 221 differentially abundant proteins were obtained. The data analysis revealed several proteins mainly related to energy metabolism, stress response and regulation of cell cycle, further analyzed by qRT-PCR, which seem important for embryogenic development. We suggest some of these proteins as key factors for the success of callus formation in oil palm including antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway. These proteins may also be potential biomarkers for the acquisition of embryogenic competence. SIGNIFICANCE: Antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway are key factors for the success of callus formation in oil palm. The proteins identified in this study may be potential biomarkers for embryogenic competence acquisition.

Insights from Proteomic Studies into Plant Somatic Embryogenesis.

Somatic embryogenesis is a biotechnological approach mainly used for the clonal propagation of different plants worldwide. In somatic embryogenesis, embryos arise from somatic cells under appropriate culture conditions. This plasticity in plants is a demonstration of true cellular totipotency and is the best approach among the genetic transformation protocols used for plant regeneration. Despite the importance of somatic embryogenesis, knowledge regarding the control of the somatic embryogenesis process is limited. Therefore, the elucidation of both the biochemical and molecular processes is important for understanding the mechanisms by which a single somatic cell becomes a whole plant. Modern proteomic techniques rely on an alternative method for the identification and quantification of proteins with different abundances in embryogenic cell cultures or somatic embryos and enable the identification of specific proteins related to somatic embryogenesis development. This review focuses on somatic embryogenesis studies that use gel-free shotgun proteomic analyses to categorize proteins that could enhance our understanding of particular aspects of the somatic embryogenesis process and identify possible targets for future studies.

Haloferax volcanii Proteome Response to Deletion of a Rhomboid Protease Gene.

Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO_1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism.