Plasmodiophora brassicae is one of the most devastating threats to Brassicaceae crops. However, the molecular mechanisms underlying clubroot disease remain unclear. Initial proteomics results led us to hypothesize that HSP70 proteins regulate host-P. brassicae interactions by modulating both plant defenses and pathogen activity. Using the Arabidopsis thaliana-P. brassicae model system, we studied the role of HSP70 proteins in detail. Through a combination of proteomics and mutant phenotype analyses, we indicate that Plasmodiophora infection induces HSP70 accumulation in Arabidopsis roots, and mutations in specific HSP70 isoforms either promote (HSP70-1, HSP70-13, HSP70-14) or suppress (HSP70-5, HSP70-12) the onset of clubroot disease. Proteomic profiling of root galls showed strong correlations between infection severity and pathogen-derived HSP70 protein CEO96729. Interactomics analyses revealed that CEO96729 interacts with host proteins involved in plant response to Plasmodiophora infection, including an extracellular GDSL esterase/lipase with a putative role in long-distance signaling, and that CEO96729 forms heterodimers with host HSP70 isoforms. These findings suggest that Plasmodiophora hijacks the host chaperone machinery to facilitate infection, offering a potential explanation for the observed modulation of disease progression in HSP70 mutants. Notably, the results also point to possible intracellular interactions with key enzymes in host physiology, including catalase 2, essential for ROS metabolism, and nitrilase, critical for auxin biosynthesis and root gall formation. Collectively, our study highlights the multifaceted roles of HSP70 proteins in Plasmodiophora pathogenicity and host-pathogen interactions, providing insights into chaperone-mediated processes in plant immunity and infection dynamics.

• Klíčová slova
• clubroot disease, interactomics, plant immunity, plant‐pathogen interaction, proteomics,
• MeSH
• Arabidopsis * parazitologie   genetika   metabolismus   MeSH
• interakce hostitele a patogenu * fyziologie   MeSH
• kořeny rostlin parazitologie   metabolismus   genetika   MeSH
• mutace MeSH
• nemoci rostlin * parazitologie   imunologie   MeSH
• Plasmodiophorida * fyziologie   MeSH
• proteiny huseníčku * metabolismus   genetika   MeSH
• proteiny tepelného šoku HSP70 * metabolismus   genetika   MeSH
• proteomika MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny huseníčku * MeSH
• proteiny tepelného šoku HSP70 * MeSH

Heavy water (D2O) is scarce in nature, and despite its physical similarity to water, D2O disrupts cellular function due to the isotope effect. While microbes can survive in nearly pure D2O, eukaryotes such as Arabidopsis thaliana are more sensitive and are unable to survive higher concentrations of D2O. To explore the underlying molecular mechanisms for these differences, we conducted a comparative proteomic analysis of E. coli, S. cerevisiae, and Arabidopsis after 180 min of growth in a D2O-supplemented media. Shared adaptive mechanisms across these species were identified, including changes in ribosomal protein abundances, accumulation of chaperones, and altered metabolism of polyamines and amino acids. However, Arabidopsis exhibited unique vulnerabilities, such as a muted stress response, lack of rapid activation of reactive oxygen species metabolism, and depletion of stress phytohormone abscisic acid signaling components. Experiments with mutants show that modulating the HSP70 pool composition may promote D2O resilience. Additionally, Arabidopsis rapidly incorporated deuterium into sucrose, indicating that photosynthesis facilitates deuterium intake. These findings provide valuable insights into the molecular mechanisms that dictate differential tolerance to D2O across species and lay the groundwork for further studies on the biological effects of uncommon isotopes, with potential implications for biotechnology and environmental science.

• Klíčová slova
• HSP70, ROS metabolism, adaptation, deuterium oxide, proteome, stress response,
• Publikační typ
• časopisecké články MeSH

The reproductive stage of plant development has the most critical impact on yield. Flowering is highly sensitive to abiotic stress, and increasing temperatures and drought harm crop yields. Salicylic acid is a phytohormone that regulates flowering and promotes stress resilience in plants. However, the exact molecular mechanisms and the level of protection are far from understood and seem to be species-specific. Here, the effect of salicylic acid was tested in a field experiment with Pisum sativum exposed to heat stress. Salicylic acid was administered at two different stages of flowering, and its effect on the yield and composition of the harvested seeds was followed. Plants treated with salicylic acid produced larger seed pods, and a significant increase in dry weight was found for the plants with a delayed application of salicylic acid. The analyses of the seed proteome, lipidome, and metabolome did not show any negative impact of salicylic treatment on seed composition. Identified processes that could be responsible for the observed improvement in seed yields included an increase in polyamine biosynthesis, accumulation of storage lipids and lysophosphatidylcholines, a higher abundance of components of chromatin regulation, calmodulin-like protein, and threonine synthase, and indicated a decrease in sensitivity to abscisic acid signaling.

• Klíčová slova
• field, heat stress, lipidome, metabolome, phytohormone, proteome, seed development, stress attenuation, yield,
• MeSH
• fyziologický stres MeSH
• hrách setý * metabolismus   MeSH
• kyselina salicylová * farmakologie   metabolismus   MeSH
• rostliny metabolismus   MeSH
• semena rostlinná metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyselina salicylová * MeSH

Plants are sessile organisms forced to adapt to environmental variations recurring in a day-night cycle. Extensive research has uncovered the transcriptional control of plants' inner clock and has revealed at least some part of the intricate and elaborate regulatory mechanisms that govern plant diel responses and provide adaptation to the ever-changing environment. Here, we analyzed the proteome of the Arabidopsis thaliana mutant genotypes collected in the middle of the day and the middle of the night, including four mutants in the phytochrome (phyA, phyB, phyC, and phyD) and the circadian clock protein LHY. Our approach provided a novel insight into the diel regulations, identifying 640 significant changes in the night-day protein abundance. The comparison with previous studies confirmed that a large portion of identified proteins was a known target of diurnal regulation. However, more than 300 were novel oscillations hidden under standard growth chamber conditions or not manifested in the wild type. Our results indicated a prominent role for ROS metabolism and phytohormone cytokinin in the observed regulations, and the consecutive analyses confirmed that. The cytokinin signaling significantly increased at night, and in the mutants, the hydrogen peroxide content was lower, and the night-day variation seemed to be lost in the phyD genotype. Furthermore, regulations in the lhy and phyB mutants were partially similar to those found in the catalase mutant cat2, indicating shared ROS-mediated signaling pathways. Our data also shed light on the role of the relatively poorly characterized Phytochrome D, pointing to its connection to glutathione metabolism and the regulation of glutathione S-transferases.

• Klíčová slova
• cytokinin, diurnal, glutathione metabolism, light, peroxide, phytochrome, signaling,
• MeSH
• apoproteiny metabolismus   MeSH
• Arabidopsis * metabolismus   MeSH
• cytokininy metabolismus   MeSH
• fytochrom B metabolismus   MeSH
• fytochrom * genetika   metabolismus   MeSH
• glutathion metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• apoproteiny MeSH
• cytokininy MeSH
• fytochrom B MeSH
• fytochrom * MeSH
• glutathion MeSH
• PHYD protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku * MeSH
• proteom MeSH
• reaktivní formy kyslíku MeSH

Poplars are among the fastest-growing trees and significant resources in agriculture and forestry. However, rapid growth requires a large water consumption, and irrigation water provides a natural means for pathogen spread. That includes members of Phytophthora spp. that have proven to be a global enemy to forests. With the known adaptability to new hosts, it is only a matter of time for more aggressive Phytophthora species to become a threat to poplar forests and plantations. Here, the effects of artificial inoculation with two different representatives of aggressive species (P. cactorum and P. plurivora) were analyzed in the proteome of the Phytophthora-tolerant hybrid poplar clone T-14 [Populus tremula L. 70 × (Populus × canescens (Ait.) Sm. 23)]. Wood microcore samples were collected at the active necrosis borders to provide insight into the molecular processes underlying the observed tolerance to Phytophthora. The analysis revealed the impact of Phytophthora on poplar primary and secondary metabolism, including carbohydrate-active enzymes, amino acid biosynthesis, phenolic metabolism, and lipid metabolism, all of which were confirmed by consecutive metabolome and lipidome profiling. Modulations of enzymes indicating systemic response were confirmed by the analysis of leaf proteome, and sampling of wood microcores in distal locations revealed proteins with abundance correlating with proximity to the infection, including germin-like proteins, components of proteosynthesis, glutamate carboxypeptidase, and an enzyme that likely promotes anthocyanin stability. Finally, the identified Phytophthora-responsive proteins were compared to those previously found in trees with compromised defense against Phytophthora, namely, Quercus spp. and Castanea sativa. That provided a subset of candidate markers of Phytophthora tolerance, including certain ribosomal proteins, auxin metabolism enzymes, dioxygenases, polyphenol oxidases, trehalose-phosphate synthase, mannose-1-phosphate guanylyltransferase, and rhamnose biosynthetic enzymes. In summary, this analysis provided the first insight into the molecular mechanisms of hybrid poplar defense against Phytophthora and identified prospective targets for improving Phytophthora tolerance in trees.

• Klíčová slova
• Phytophthora cactorum, Phytophthora plurivora, Populus, biotic interaction, lipidome, metabolome, proteome,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Split-root systems (SRS) have many applications in plant sciences, but their implementation, depending on the experimental design, can be difficult and time-consuming. Additionally, the system is not exempt from limitations, since the time required for the establishment of the SRS imposes a limit to how early in plant development experiments can be performed. Here, we optimized and explained in detail a method for establishing a SRS in young Arabidopsis thaliana seedlings, both in vitro and in soil. RESULTS: We found that the partial de-rooting minimized the recovery time compared to total de-rooting, thus allowing the establishment of the split-root system in younger plants. Analysis of changes in the Arabidopsis leaf proteome following the de-rooting procedure highlighted the distinct metabolic alterations that totally and partially de-rooted plants undergo during the healing process. This system was also validated for its use in drought experiments, as it offers a way to apply water-soluble compounds to plants subjected to drought stress. By growing plants in a split-root system with both halves being water-deprived, it is possible to apply the required compound to one half of the root system, which can be cut from the main plant once the compound has been absorbed, thus minimizing rehydration and maintaining drought conditions. CONCLUSIONS: Partial de-rooting is the suggested method for obtaining split-root systems in small plants like Arabidopsis thaliana, as growth parameters, survival rate, and proteomic analysis suggest that is a less stressful procedure than total de-rooting, leading to a final rosette area much closer to that of uncut plants. Additionally, we provide evidence that split root-systems can be used in drought experiments where water-soluble compounds are applied with minimal effects of rehydration.

• Klíčová slova
• Arabidopsis thaliana, Drought stress, Phytohormones, Proteomics, Split-root systems,
• Publikační typ
• časopisecké články MeSH

Phytophthora is arguably one of the most damaging genera of plant pathogens. This pathogen is well suited to transmission via the international plant trade, and globalization has been promoting its spread since the 19th century. Early detection is essential for reducing its economic and ecological impact. Here, a shotgun proteomics approach was utilized for Phytophthora analysis. The collection of 37 Phytophthora isolates representing 12 different species was screened for species-specific peptide patterns. Next, Phytophthora proteins were detected in planta, employing model plants Solanum tuberosum and Hordeum vulgare. Although the evolutionarily conserved sequences represented more than 10% of the host proteome and limited the pathogen detection, the comparison between qPCR and protein data highlighted more than 300 protein markers, which correlated positively with the amount of P. infestans DNA. Finally, the analysis of P. palmivora response in barley revealed significant alterations in plant metabolism. These changes included enzymes of cell wall metabolism, ROS production, and proteins involved in trafficking. The observed root-specific attenuation in stress-response mechanisms, including the biosynthesis of jasmonates, ethylene and polyamines, and an accumulation of serotonin, provided the first insight into molecular mechanisms behind this particular biotic interaction.

• Klíčová slova
• Hordeum vulgare, P. infestans, P. palmivora, Phytophthora, Solanum tuberosum, leaf inoculation, proteomics,
• MeSH
• chromatografie kapalinová MeSH
• fyziologický stres MeSH
• hmotnostní spektrometrie MeSH
• ječmen (rod) enzymologie   metabolismus   mikrobiologie   MeSH
• listy rostlin metabolismus   mikrobiologie   MeSH
• metabolické sítě a dráhy MeSH
• nemoci rostlin mikrobiologie   MeSH
• peptidy metabolismus   MeSH
• Phytophthora infestans genetika   izolace a purifikace   MeSH
• polymerázová řetězová reakce MeSH
• proteom metabolismus   MeSH
• proteomika MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• Solanum tuberosum metabolismus   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy MeSH
• proteom MeSH
• reaktivní formy kyslíku MeSH
• rostlinné proteiny MeSH

Cytokinin is a phytohormone involved in the regulation of diverse developmental and physiological processes in plants. Its potential in biotechnology and for development of higher-yield and more resilient plants has been recognized, yet the molecular mechanisms behind its action are far from understood. In this report, the roots of barley seedlings were explored as a new source to reveal as yet unknown cytokinin-responsive proteins for crop improvement. Here we found significant differences reproducibly observed for 178 proteins, for which some of the revealed cytokinin-responsive pathways were confirmed in metabolome analysis, including alterations phenylpropanoid pathway, amino acid biosynthesis and ROS metabolism. Bioinformatics analysis indicated a significant overlap between cytokinin response and response to abiotic stress. This was confirmed by comparing proteome and metabolome profiles in response to drought, salinity or a period of temperature stress. The results illustrate complex abiotic stress response in the early development of model crop plant and confirm an extensive crosstalk between plant hormone cytokinin and response to temperature stimuli, water availability or salinity stress.

• Klíčová slova
• Hordeum vulgare, ROS, abiotic stress, metabolome, phenylpropanoid biosynthesis, proteome, root, zeatin,
• Publikační typ
• časopisecké články MeSH