Proteome- and metabolome-level changes during early stages of clubroot infection in Brassica napus canola.

Clubroot is a destructive root disease of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin. Despite extensive research into the molecular responses of B. napus to P. brassicae, there is limited information on proteome- and metabolome-level changes in response to the pathogen, especially during the initial stages of infection. In this study, we have investigated the proteome- and metabolome- level changes in the roots of clubroot-resistant (CR) and -susceptible (CS) doubled-haploid (DH) B. napus lines, in response to P. brassicae pathotype 3H at 1-, 4-, and 7-days post-inoculation (DPI). Root proteomes were analyzed using nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS). Comparisons of pathogen-inoculated and uninoculated root proteomes revealed 2515 and 1556 differentially abundant proteins at one or more time points (1-, 4-, and 7-DPI) in the CR and CS genotypes, respectively. Several proteins related to primary metabolites (e.g., amino acids, fatty acids, and lipids), secondary metabolites (e.g., glucosinolates), and cell wall reinforcement-related proteins [e.g., laccase, peroxidases, and plant invertase/pectin methylesterase inhibitors (PInv/PMEI)] were identified. Eleven nucleotides and nucleoside-related metabolites, and eight fatty acids and sphingolipid-related metabolites were identified in the metabolomics study. To our knowledge, this is the first report of root proteome-level changes and associated alterations in metabolites during the early stages of P. brassicae infection in B. napus.

Multi-omics insights into the positive role of strigolactone perception in barley drought response.

BACKGROUND: Drought is a major environmental stress that affects crop productivity worldwide. Although previous research demonstrated links between strigolactones (SLs) and drought, here we used barley (Hordeum vulgare) SL-insensitive mutant hvd14 (dwarf14) to scrutinize the SL-dependent mechanisms associated with water deficit response. RESULTS: We have employed a combination of transcriptomics, proteomics, phytohormonomics analyses, and physiological data to unravel differences between wild-type and hvd14 plants under drought. Our research revealed that drought sensitivity of hvd14 is related to weaker induction of abscisic acid-responsive genes/proteins, lower jasmonic acid content, higher reactive oxygen species content, and lower wax biosynthetic and deposition mechanisms than wild-type plants. In addition, we identified a set of transcription factors (TFs) that are exclusively drought-induced in the wild-type barley. CONCLUSIONS: Critically, we resolved a comprehensive series of interactions between the drought-induced barley transcriptome and proteome responses, allowing us to understand the profound effects of SLs in alleviating water-limiting conditions. Several new avenues have opened for developing barley more resilient to drought through the information provided. Moreover, our study contributes to a better understanding of the complex interplay between genes, proteins, and hormones in response to drought, and underscores the importance of a multidisciplinary approach to studying plant stress response mechanisms.

Systems-level proteomics and metabolomics reveals the diel molecular landscape of diverse kale cultivars.

Kale is a group of diverse Brassicaceae species that are nutritious leafy greens consumed for their abundance of vitamins and micronutrients. Typified by their curly, serrated and/or wavy leaves, kale varieties have been primarily defined based on their leaf morphology and geographic origin, despite having complex genetic backgrounds. Kale is a very promising crop for vertical farming due to its high nutritional content; however, being a non-model organism, foundational, systems-level analyses of kale are lacking. Previous studies in kale have shown that time-of-day harvesting can affect its nutritional composition. Therefore, to gain a systems-level diel understanding of kale across its wide-ranging and diverse genetic landscape, we selected nine publicly available and commercially grown kale cultivars for growth under near-sunlight LED light conditions ideal for vertical farming. We then analyzed changes in morphology, growth and nutrition using a combination of plant phenotyping, proteomics and metabolomics. As the diel molecular activities of plants drive their daily growth and development, ultimately determining their productivity as a crop, we harvested kale leaf tissue at both end-of-day (ED) and end-of-night (EN) time-points for all molecular analyses. Our results reveal that diel proteome and metabolome signatures divide the selected kale cultivars into two groups defined by their amino acid and sugar content, along with significant proteome differences involving carbon and nitrogen metabolism, mRNA splicing, protein translation and light harvesting. Together, our multi-cultivar, multi-omic analysis provides new insights into the molecular underpinnings of the diel growth and development landscape of kale, advancing our fundamental understanding of this nutritious leafy green super-food for horticulture/vertical farming applications.

Substrate profiling of the Arabidopsis Ca2+-dependent protein kinase AtCPK4 and its Ricinus communis ortholog RcCDPK1.

AtCPK4 and AtCPK11 are Arabidopsis thaliana Ca2+-dependent protein kinase (CDPK) paralogs that have been reported to positively regulate abscisic acid (ABA) signal transduction by phosphorylating ABA-responsive transcription factor-4 (AtABF4). By contrast, RcCDPK1, their closest Ricinus communis ortholog, participates in the control of anaplerotic carbon flux in developing castor oil seeds by catalyzing inhibitory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser451. LC-MS/MS revealed that AtCPK4 and RcCDPK1 transphosphorylated several common, conserved residues of AtABF4 and its castor ortholog, TRANSCRIPTION FACTOR RESPONSIBLE FOR ABA REGULATON. Arabidopsis atcpk4/atcpk11 mutants displayed an ABA-insensitive phenotype that corroborated the involvement of AtCPK4/11 in ABA signaling. A kinase-client assay was employed to identify additional AtCPK4/RcCDPK1 targets. Both CDPKs were separately incubated with a library of 2095 peptides representative of Arabidopsis protein phosphosites; five overlapping targets were identified including PLANT INTRACELLULAR RAS-GROUP-RELATED LEUCINE-RICH REPEAT PROTEIN-9 (AtPIRL9) and the E3-ubiquitin ligase ARABIDOPSIS TOXICOS EN LEVADURA 6 (AtATL6). AtPIRL9 and AtATL6 residues phosphorylated by AtCPK4/RcCDPK1 conformed to a CDPK recognition motif that was conserved amongst their respective orthologs. Collectively, this study provides evidence for novel AtCPK4/RcCDPK1 substrates, which may help to expand regulatory networks linked to Ca2+- and ABA-signaling, immune responses, and central carbon metabolism.

Persisting Effects in Daphnia magna Following an Acute Exposure to Flowback and Produced Waters from the Montney Formation.

Hydraulic fracturing extracts oil and gas through the injection of water and proppants into subterranean formations. These injected fluids mix with the host rock formation and return to the surface as a complex wastewater containing salts, metals, and organic compounds, termed flowback and produced water (FPW). Previous research indicates that FPW is toxic to Daphnia magna (D. magna), impairing reproduction, molting, and maturation time; however, recovery from FPW has not been extensively studied. Species unable to recover have drastic impacts on populations on the ecological scale; thus, this study sought to understand if recovery from an acute 48 h FPW exposure was possible in the freshwater invertebrate, D. magna by using a combination of physiological and molecular analyses. FPW (0.75%) reduced reproduction by 30% and survivorship to 32% compared to controls. System-level quantitative proteomic analyses demonstrate extensive perturbation of metabolism and protein transport in both 0.25 and 0.75% FPW treatments after a 48 h FPW exposure. Collectively, our data indicate that D. magna are unable to recover from acute 48 h exposures to ≥0.25% FPW, as evidence of toxicity persists for at least 19 days post-exposure. This study highlights the importance of considering persisting effects following FPW remediation when modeling potential spill scenarios.

eLife's new model and its impact on science communication.

The eLife Early-Career Advisory Group discusses eLife's new peer review and publishing model, and how the whole process of scientific communication could be improved for the benefit of early-career researchers and the entire scientific community.

Recommendations for empowering early career researchers to improve research culture and practice.

Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice; and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and examples of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian.

Multi-omic analysis shows REVEILLE clock genes are involved in carbohydrate metabolism and proteasome function.

Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE (RVE) proteins are recently discovered members of the plant circadian circuitry that activate the evening complex and PSEUDO-RESPONSE REGULATOR genes to maintain regular circadian oscillation. The RVE8 protein and its two homologs, RVE 4 and 6 in Arabidopsis (Arabidopsis thaliana), have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass, cell size, and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we draw new connections between RVE8-like proteins and a number of core plant cell processes. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid, and lipid metabolism, including a starch excess phenotype at dawn. We further demonstrate that rve 4 6 8 plants have lower levels of 20S proteasome subunits and possess significantly reduced proteasome activity, potentially explaining the increase in cell-size observed in RVE8-like mutants. Overall, this robust, multi-omic dataset provides substantial insight into the far-reaching impact RVE8-like proteins have on the diel plant cell environment.

A Proteome-Level Investigation Into Plasmodiophora brassicae Resistance in Brassica napus Canola.

Clubroot of Brassicaceae, an economically important soil borne disease, is caused by Plasmodiophora brassicae Woronin, an obligate, biotrophic protist. This disease poses a serious threat to canola and related crops in Canada and around the globe causing significant losses. The pathogen is continuously evolving and new pathotypes are emerging, which necessitates the development of novel resistant canola cultivars to manage the disease. Proteins play a crucial role in many biological functions and the identification of differentially abundant proteins (DAP) using proteomics is a suitable approach to understand plant-pathogen interactions to assist in the development of gene specific markers for developing clubroot resistant (CR) cultivars. In this study, P. brassicae pathotype 3 (P3H) was used to challenge CR and clubroot susceptible (CS) canola lines. Root samples were collected at three distinct stages of pathogenesis, 7-, 14-, and 21-days post inoculation (DPI), protein samples were isolated, digested with trypsin and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. A total of 937 proteins demonstrated a significant (q-value < 0.05) change in abundance in at least in one of the time points when compared between control and inoculated CR-parent, CR-progeny, CS-parent, CS-progeny and 784 proteins were significantly (q < 0.05) changed in abundance in at least in one of the time points when compared between the inoculated- CR and CS root proteomes of parent and progeny across the three time points tested. Functional annotation of differentially abundant proteins (DAPs) revealed several proteins related to calcium dependent signaling pathways. In addition, proteins related to reactive oxygen species (ROS) biochemistry, dehydrins, lignin, thaumatin, and phytohormones were identified. Among the DAPs, 73 putative proteins orthologous to CR proteins and quantitative trait loci (QTL) associated with eight CR loci in different chromosomes including chromosomes A3 and A8 were identified. Proteins including BnaA02T0335400WE, BnaA03T0374600WE, BnaA03T0262200WE, and BnaA03T0464700WE are orthologous to identified CR loci with possible roles in mediating clubroot responses. In conclusion, these results have contributed to an improved understanding of the mechanisms involved in mediating response to P. brassicae in canola at the protein level.

BoxCar and Library-Free Data-Independent Acquisition Substantially Improve the Depth, Range, and Completeness of Label-Free Quantitative Proteomics.

Data-dependent acquisition (DDA) methods are the current standard for quantitative proteomics in many biological systems. However, DDA preferentially measures highly abundant proteins and generates data that is plagued with missing values, requiring extensive imputation. Here, we demonstrate that library-free BoxCarDIA acquisition, combining MS1-level BoxCar acquisition with MS2-level data-independent acquisition (DIA) analysis, outperforms conventional DDA and other library-free DIA (directDIA) approaches. Using a combination of low- (HeLa cells) and high- (Arabidopsis thaliana cell culture) dynamic range sample types, we demonstrate that BoxCarDIA can achieve a 40% increase in protein quantification over DDA without offline fractionation or an increase in mass-spectrometer acquisition time. Further, we provide empirical evidence for substantial gains in dynamic range sampling that translates to deeper quantification of low-abundance protein classes under-represented in DDA and directDIA data. Unlike both DDA and directDIA, our new BoxCarDIA method does not require full MS1 scans while offering reproducible protein quantification between replicate injections and providing more robust biological inferences. Overall, our results advance the BoxCarDIA technique and establish it as the new method of choice for label-free quantitative proteomics across diverse sample types.

Quantitative Proteome and PTMome Analysis of Arabidopsis thaliana Root Responses to Persistent Osmotic and Salinity Stress.

Abiotic stresses such as drought result in large annual economic losses around the world. As sessile organisms, plants cannot escape the environmental stresses they encounter but instead must adapt to survive. Studies investigating plant responses to osmotic and/or salt stress have largely focused on short-term systemic responses, leaving our understanding of intermediate to longer-term adaptation (24 h to d) lacking. In addition to protein abundance and phosphorylation changes, evidence suggests reversible lysine acetylation may also be important for abiotic stress responses. Therefore, to characterize the protein-level effects of osmotic and salt stress, we undertook a label-free proteomic analysis of Arabidopsis thaliana roots exposed to 300 mM mannitol and 150 mM NaCl for 24 h. We assessed protein phosphorylation, lysine acetylation and changes in protein abundance, detecting significant changes in 245, 35 and 107 total proteins, respectively. Comparison with available transcriptome data indicates that transcriptome- and proteome-level changes occur in parallel, while post-translational modifications (PTMs) do not. Further, we find significant changes in PTMs, and protein abundance involve different proteins from the same networks, indicating a multifaceted regulatory approach to prolonged osmotic and salt stress. In particular, we find extensive protein-level changes involving sulfur metabolism under both osmotic and salt conditions as well as changes in protein kinases and transcription factors that may represent new targets for drought stress signaling. Collectively, we find that protein-level changes continue to occur in plant roots 24 h from the onset of osmotic and salt stress and that these changes differ across multiple proteome levels.

DomainViz: intuitive visualization of consensus domain distributions across groups of proteins.

The prediction of functional domains is typically among the first steps towards understanding the function of new proteins and protein families. There are numerous databases of annotated protein domains that permit researchers to identify domains on individual proteins of interest. However, it is necessary to perform high-throughput domain searches to gain evolutionary insight into the functions of proteins and protein families. Unfortunately, at present, it is difficult to search for, and visualize domain conservation across multiple proteins and/or multiple groups of proteins in an intuitive manner. Here we present DomainViz, a new web-server that streamlines the identification and visualization of domains across multiple protein sequences. Currently, DomainViz uses the well-established PFAM and Prosite databases for domain searching and assembles intuitive, publication-ready 'monument valley' plots (mv-plots) that display the extent of domain conservation along two dimensions: positionality and frequency of occurrence in the input protein sequences. In addition, DomainViz produces a conventional domain-ordering figure. DomainViz can be used to explore the conservation of domains within a single protein family, across multiple families, and across families from different species to support studies into protein function and evolution. The web-server is publicly available at: https://uhrigprotools.biology.ualberta.ca/domainviz.

Closing the protein gap in plant chronobiology.

Our modern understanding of diel cell regulation in plants stems from foundational work in the late 1990s that analysed the dynamics of selected genes and mutants in Arabidopsis thaliana. The subsequent rise of transcriptomics technologies such as microarrays and RNA sequencing has substantially increased our understanding of anticipatory (circadian) and reactive (light- or dark-triggered) diel events in plants. However, it is also becoming clear that gene expression data fail to capture critical events in diel regulation that can only be explained by studying protein-level dynamics. Over the past decade, mass spectrometry technologies and quantitative proteomic workflows have significantly advanced, finally allowing scientists to characterise diel protein regulation at high throughput. Initial proteomic investigations suggest that the diel transcriptome and proteome generally lack synchrony and that the timing of daily regulatory events in plants is impacted by multiple levels of protein regulation (e.g., post-translational modifications [PTMs] and protein-protein interactions [PPIs]). Here, we highlight and summarise how the use of quantitative proteomics to elucidate diel plant cell regulation has advanced our understanding of these processes. We argue that this new understanding, coupled with the extraordinary developments in mass spectrometry technologies, demands greater focus on protein-level regulation of, and by, the circadian clock. This includes hitherto unexplored diel dynamics of protein turnover, PTMs, protein subcellular localisation and PPIs that can be masked by simple transcript- and protein-level changes. Finally, we propose new directions for how the latest advancements in quantitative proteomics can be utilised to answer outstanding questions in plant chronobiology.

Phosphate and phosphite have a differential impact on the proteome and phosphoproteome of Arabidopsis suspension cell cultures.

Phosphorus absorbed in the form of phosphate (H2 PO4- ) is an essential but limiting macronutrient for plant growth and agricultural productivity. A comprehensive understanding of how plants respond to phosphate starvation is essential for the development of more phosphate-efficient crops. Here we employed label-free proteomics and phosphoproteomics to quantify protein-level responses to 48 h of phosphate versus phosphite (H2 PO3- ) resupply to phosphate-deprived Arabidopsis thaliana suspension cells. Phosphite is similarly sensed, taken up and transported by plant cells as phosphate, but cannot be metabolized or used as a nutrient. Phosphite is thus a useful tool for differentiating between non-specific processes related to phosphate sensing and transport and specific responses to phosphorus nutrition. We found that responses to phosphate versus phosphite resupply occurred mainly at the level of protein phosphorylation, complemented by limited changes in protein abundance, primarily in protein translation, phosphate transport and scavenging, and central metabolism proteins. Altered phosphorylation of proteins involved in core processes such as translation, RNA splicing and kinase signaling was especially important. We also found differential phosphorylation in response to phosphate and phosphite in 69 proteins, including splicing factors, translation factors, the PHT1;4 phosphate transporter and the HAT1 histone acetyltransferase - potential phospho-switches signaling changes in phosphorus nutrition. Our study illuminates several new aspects of the phosphate starvation response and identifies important targets for further investigation and potential crop improvement.

Twelve Principles Trainees, PIs, Departments, and Faculties Can Use to Reduce Bias and Discrimination in STEM.

There is an overwhelming amount of evidence demonstrating that people from marginalized groups, including women, racialized and Indigenous peoples, people with disabilities, immigrants, and LGBTQ+ individuals, continue to face substantial discrimination in STEM, manifested as both overt bias and unconscious bias. These biases result in discrimination against individuals in marginalized groups, and independent biases collectively contribute to a culture that systematically discriminates against people from marginalized groups. Representation from marginalized groups in postsecondary degrees in natural science and engineering has not substantially improved in over a decade. A set of 10 concrete principles are presented that trainees, principle investigators, departments, and faculties can use to enhance the participation and lived experiences of people in marginalized groups in STEM.