Plant Proteomic Research 4.0

As an important tool of systems biology, proteomics has enabled a deep understanding of different plant processes and functions. Complemented with genomic data, computational tools, and improved sample preparation strategies, proteomics has an unprecedented opportunity to characterize plant proteoforms in high spatial and temporal resolution. This special issue of Plant Proteomic Research 4.0 captures the recent advancements in proteomics and addresses the current challenges of plant stress response and resilience in the ever-changing climate. It contains 12 articles, including three reviews and nine original research articles. The three reviews deal with pollen phosphoproteomics, starch biosynthesis-related proteins and posttranslational modifications (PTMs) in rice developing seeds, and PTMs of waxy proteins in rice grain. The nine research articles include three related to temperature, two on water stress, two on salt stress, one on fungal pathogen, and the last one on field-grown potato apoplast proteome. The articles reflect the current frontiers of plant proteomics, focusing on themes of environmental stresses, proteoforms/PTMs, crop species, and new development in data-independent acquisition mass spectrometry. They provide readers insights into current technologies, their utility in understanding plant growth and resilience, as well as directions of proteomics in the frontiers of systems biology and synthetic biology

Molecular and Genetic Analysis of the effects of SUMOylation on the regulation of floral transition in Arabidopsis

SUMOylation, the post-translational attachment of SUMO (Small Ubiquitin-like Modifier) to a substrate protein, regulates the activity of several proteins involved in critical cellular processes like cell division and transcriptional regulation. SUMO is subsequently removed from substrates by SUMO-specific proteases, making this modification reversible. In plants, SUMOylation has been implicated in several physiological responses and flowering time control. ESD4 (Early in Short Days 4) encodes a SUMO-specific protease that prevents the accumulation of SUMO-conjugates in Arabidopsis. The esd4-1 mutant shows a very early flowering phenotype as well as several shoot developmental distortions suggesting an important role of SUMOylation in the regulation of plant development. To investigate the role of SUMOylation in flowering time control a suppressor screen of esd4 was performed. 120 independent suppressors of esd4 (sed) were isolated and 15 of them further characterized. The SUMO-conjugate levels of these seds are more similar to those of esd4-1 than to the wild type. Rough map positions for five of these sed mutants were established using classical genetic methods, and combined with Next Generation Sequencing sed111-1 was finemapped to a region of chromosome I that contains only six candidate genes. In a different study, SUMOylation of SHORT VEGETATIVE PHASE (SVP) was assessed and SUMO attachment lysines were determined using E. coli strains that recapitulate the SUMO conjugation pathway. SVP interacts with FLOWERING LOCUS C (FLC) to form a strong floral repressor complex. To study the role of SUMOylation in SVP function, an svp-null mutant (svp-41) was transformed with constructs aiming to hyperSUMOylate (translational fusions with SUMO or AtSCE) or hypoSUMOylate (mutations in the putative SUMO-attachment sites) the SVP protein in transgenic plants. Mutant phenotypes caused by these constructs are discussed

Evidence for a strong correlation between transcription factor protein disorder and organismic complexity

Studies of diverse phylogenetic lineages reveal that protein disorder increases in concert with organismic complexity but that differences nevertheless exist among lineages. To gain insight into this phenomenology, we analyzed all of the transcription factor (TF) families for which sequences are known for 17 species spanning bacteria, yeast, algae, land plants, and animals and for which the number of different cell types has been reported in the primary literature. Although the fraction of disordered residues in TF sequences is often moderately or poorly correlated with organismic complexity as gauged by cell-type number (r20.8). Furthermore, the correlation between the fraction of disordered residues and cell-type number becomes stronger when confined to the TF families participating in cell cycle, cell size, cell division, cell differentiation, or cell proliferation, and other important developmental processes. The data also indicate that evolutionarily simpler organisms allow for the detection of subtle differences in the conserved IDRs of TFs as well as changes in variable IDRs, which can influence the DNA recognition and multifunctionality of TFs through direct or indirect mechanisms. Although strong correlations cannot be taken as evidence for causeand-effect relationships, we interpret our data to indicate that increasing TF disorder likely was an important factor contributing to the evolution of organismic complexity and not merely a concurrent unrelated effect of increasing organismic complexity

pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model

BACKGROUND: Protein kinase A (cAMP-dependent kinase, PKA) is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal. RESULTS: Approximately 20 sequence positions flanking the phosphorylated residue on both sides have been found to be restricted in their sequence variability (region -18...+23 with the site at position 0). The conserved physical pattern can be rationalized in terms of a qualitative binding model with the catalytic cleft of the protein kinase A. Positions -6...+4 surrounding the phosphorylation site are influenced by direct interaction with the kinase in a varying degree. This sequence stretch is embedded in an intrinsically disordered region composed preferentially of hydrophilic residues with flexible backbone and small side chain. This knowledge has been incorporated into a simplified analytical model of productive binding of substrate proteins with PKA. CONCLUSION: The scoring function of the pkaPS predictor can confidently discriminate PKA phosphorylation sites from serines/threonines with non-permissive sequence environments (sensitivity of ~96% at a specificity of ~94%). The tool "pkaPS" has been applied on the whole human proteome. Among new predicted PKA targets, there are entirely uncharacterized protein groups as well as apparently well-known families such as those of the ribosomal proteins L21e, L22 and L6. AVAILABILITY: The supplementary data as well as the prediction tool as WWW server are available at . REVIEWERS: Erik van Nimwegen (Biozentrum, University of Basel, Switzerland), Sandor Pongor (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy), Igor Zhulin (University of Tennessee, Oak Ridge National Laboratory, USA)

Occurrence of Hydroxyproline in Proteomes of Higher Plants

Food allergies affect millions of individuals across the United States and worldwide. Peanut allergies are among the most severe food allergies because of their potentially life-threatening symptoms and lifelong persistence. Potent peanut allergen, Ara h 2, is known to contain an amino acid motif containing the posttranslational modification, hydroxyproline (HyP). HyP is associated with immunogenic response when present both in Ara h 2 and in timothy grass pollen allergen, Phl p 1. To further explore the presence of HyP in higher plants and specifically to investigate its potential presence in commonly allergenic plants, a study of 26 plant seeds was conducted using hydrolyzed amino acid analysis (HAA) and data-dependent acquisition (DDA) through liquid chromatography and tandem mass spectrometry (LCMS/MS). Curated protein databases allowed for database searches using PEAKS software. Samples for which no database could be procured were analyzed using de novo sequencing. Results showed detection of HyP in 25 out of 26 plant seed samples. HyP sites were classified into one of four tiers based on the quality of the database used to identify a given site. To further refine the identified HyP sites and to increase confidence in their position and identity, a manual analysis approach was performed in addition to software analyses. This approach was successful in reducing the number of sites for each sample as well as increasing the confidence of those sites. Peanut presented as a clear outlier in the number of HyP sites in software and de novo analyses both before and after refinement by manual analysis. The results indicate that species across Viridiplantae possess the machinery to perform prolyl hydroxylation. Furthermore, these data indicate that peanut is unique in its quantity of HyP sites even when normalized to the total number of proline residues. Advisors: Joseph L. Baumert and Philip Johnso

The evolution, modifications and interactions of proteins and RNAs

Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism

Study of C-Repeat Binding Factors (CBF) structural elements conferring stability under adverse conditions

Winter wheat is a crop with a lot of potential for the Canadian Prairies. It has many agronomic, environmental and economic advantages over spring wheat, such as a 20-30 % higher yield potential and a lower rate of Fusarium infections. However, wheat production on the Prairies is dominated by spring varieties because the current winter wheat cultivars do not accumulate sufficient frost resistance during cold acclimation in the autumn to survive the winters on a consistent basis. Efforts to improve frost tolerance genes in winter wheat via conventional breeding practices has been attempted but proven to be difficult as the cold tolerance in the available varieties cannot be exceeded. A possible approach to increase frost hardiness in plants is through the optimization of CRT (C-repeat)-Binding Factor (CBF) expression, activities and stability under cold conditions. These master transcription factors upregulate a wide range of cold stress responses and represent about 40% of the difference in freezing tolerance observed between Canadian winter wheat cultivars with low and very high winter-hardiness. Cold-hardy winter wheat produces many CBF variants, but little information is available about the role of the individual proteins. From initial studies of the CBF family encoded by cold-hardy cultivar Norstar, several members display high resilience to extreme temperatures and denaturing conditions, which may be important for winter survival. The high CBF stability is also associated with anomalous slow migration on SDS-PAGE gels. To further investigate the abnormal gel behavior of CBF12, various modifications to the protein were made and analyzed by SDS-PAGE. It was concluded that the conserved CMIII3, CMIII-1 motifs flanking the DNA-binding AP2 domain or the AP2 domain itself do not slow down CBF12 migration during SDS-PAGE. Rather, removal of the secondary structures within the AP2 region increased the degree of anomalous migration. Instead, it was found that changes in the ratio and distribution of charged residues and the degree of disorder predicted for CBF12 correlated with increased anomalous migration during SDS-PAGE. Similar features are also present for other stress response proteins and transcription factors. Whether the specific amino acid residues contributing to gel shifting play a role in enhancing cold tolerance in planta remains to be verified

Killing The Messenger: Exploring Novel Triggers For Messenger Rna Decay In Eukaryotes

The lifecycle of messenger RNAs is regulated by multiple layers beyond their primary sequence. In addition to carrying the information for protein synthesis, mRNAs are decorated with RNA binding proteins, marked with covalent chemical modifications, and fold into intricate secondary structures. However, the full set of information encoded by these “epitranscriptomic” layers is only partially understood, and is often only characterized for select transcripts. Thus, it is crucial to develop and apply transcriptome-wide analytical tools to probe the location and functional relevance of epitranscriptome features. In this dissertation, I focus on applying such methods toward better understanding determinants of mRNA stability, through using 1) High Throughput Annotation of Modified Nucleotides, 2) nuclease-mediated probing of RNA secondary structure, and 3) detection of partial mRNA degradation from RNA sequencing. I observe that chemical modifications tend to mark uncapped and small RNA fragments derived from mRNAs in plants and humans, suggesting a link between modifications and mRNA stability. I then show this link is direct through showing differential stability at Arabidopsis transcripts that change modification status during long-term salt stress. By probing secondary structure, I show a link between structure, smRNA production, and co-translational RNA decay. Finally, I develop a new in silico method to detect partial RNA degradation in mouse oocytes, and identify sequence elements that appear to block complete exonucleolytic transcript cleavage during meiosis. I then identify putative RNA binding proteins that might mediate this partial decay. In summary, I apply transcriptome-wide sequencing-based methods to survey the effects of covalent modifications, secondary structure, and RNA binding proteins on mRNA stability