The fungus Acremonium alternatum enhances salt stress tolerance by regulating host redox homeostasis and phytohormone signaling.

While endophytic fungi offer promising avenues for bolstering plant resilience against abiotic stressors, the molecular mechanisms behind this biofortification remain largely unknown. This study employed a multifaceted approach, combining plant physiology, proteomic, metabolomic, and targeted hormonal analyses to illuminate the early response of Brassica napus to Acremonium alternatum during the nascent stages of their interaction. Notably, under optimal growth conditions, the initial reaction to fungus was relatively subtle, with no visible alterations in plant phenotype and only minor impacts on the proteome and metabolome. Interestingly, the identified proteins associated with the Acremonium response included TUDOR 1, Annexin D4, and a plastidic K+ efflux antiporter, hinting at potential processes that could counter abiotic stressors, particularly salt stress. Subsequent experiments validated this hypothesis, showcasing significantly enhanced growth in Acremonium-inoculated plants under salt stress. Molecular analyses revealed a profound impact on the plant's proteome, with over 50% of salt stress response proteins remaining unaffected in inoculated plants. Acremonium modulated ribosomal proteins, increased abundance of photosynthetic proteins, enhanced ROS metabolism, accumulation of V-ATPase, altered abundances of various metabolic enzymes, and possibly promoted abscisic acid signaling. Subsequent analyses validated the accumulation of this hormone and its enhanced signaling. Collectively, these findings indicate that Acremonium promotes salt tolerance by orchestrating abscisic acid signaling, priming the plant's antioxidant system, as evidenced by the accumulation of ROS-scavenging metabolites and alterations in ROS metabolism, leading to lowered ROS levels and enhanced photosynthesis. Additionally, it modulates ion sequestration through V-ATPase accumulation, potentially contributing to the observed decrease in chloride content.

Abiotic Stress in Crop Production.

The vast majority of agricultural land undergoes abiotic stress that can significantly reduce agricultural yields. Understanding the mechanisms of plant defenses against stresses and putting this knowledge into practice is, therefore, an integral part of sustainable agriculture. In this review, we focus on current findings in plant resistance to four cardinal abiotic stressors-drought, heat, salinity, and low temperatures. Apart from the description of the newly discovered mechanisms of signaling and resistance to abiotic stress, this review also focuses on the importance of primary and secondary metabolites, including carbohydrates, amino acids, phenolics, and phytohormones. A meta-analysis of transcriptomic studies concerning the model plant Arabidopsis demonstrates the long-observed phenomenon that abiotic stressors induce different signals and effects at the level of gene expression, but genes whose regulation is similar under most stressors can still be traced. The analysis further reveals the transcriptional modulation of Golgi-targeted proteins in response to heat stress. Our analysis also highlights several genes that are similarly regulated under all stress conditions. These genes support the central role of phytohormones in the abiotic stress response, and the importance of some of these in plant resistance has not yet been studied. Finally, this review provides information about the response to abiotic stress in major European crop plants-wheat, sugar beet, maize, potatoes, barley, sunflowers, grapes, rapeseed, tomatoes, and apples.

Salicylic Acid Treatment and Its Effect on Seed Yield and Seed Molecular Composition of Pisum sativum under Abiotic Stress.

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.

Environmental impacts on barley grain composition and longevity.

To counter projected reductions in yields of the major crop barley, it is essential to elucidate the mechanisms of its resilience. To assist such efforts, we collected grains from plants grown in fields at 12 testing stations, with suitable temperature and precipitation gradients for identifying environmentally induced changes in their protein and metabolite contents. We then subjected the grains to detailed molecular analysis. The results showed that numerous metabolites and at least a quarter of the grain protein content was modulated by the environment, and provided insights into barley seed production under abiotic stress, including alterations in ribosomal proteins, heatshock protein 70 family proteins, inhibitors, storage proteins, and lipid droplet formation. Potential positive and negative markers of yield were also identified, including the phenolic compound catechin and storage protein levels, respectively. Complementary analyses of barley seedlings and Arabidopsis seeds, respectively, confirmed the role of the identified proteins in abiotic stress responses and highlighted evolutionarily conserved mechanisms. In addition, accelerated ageing experiments revealed that variations in the environment had stronger effects on seed longevity than the genotype. Finally, seeds with the highest longevity differed from the others in gibberellin contents, H2O2 metabolism, and levels of >250 proteins, providing novel targets for improving resilience.

Defense mechanisms promoting tolerance to aggressive Phytophthora species in hybrid poplar.

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.

Phytochromes and Their Role in Diurnal Variations of ROS Metabolism and Plant Proteome.

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.

Molecular Mechanisms Underlying Flax (Linum usitatissimum L.) Tolerance to Cadmium: A Case Study of Proteome and Metabolome of Four Different Flax Genotypes.

Cadmium is one of the most toxic heavy metal pollutants, and its accumulation in the soil is harmful to agriculture. Plants have a higher cadmium tolerance than animals, and some species can be used for phytoremediation. Flax (Linum usitatissimum L.) can accumulate high amounts of cadmium, but the molecular mechanism behind its tolerance is unknown. Here, we employed four genotypes representing two fiber cultivars, an oilseed breeding line, and a transgenic line overexpressing the metallothionein domain for improved cadmium tolerance. We analyzed the proteome of suspensions and the proteome and metabolome of seedling roots in response to cadmium. We identified more than 1400 differentially abundant proteins representing putative mechanisms in cadmium tolerance, including metal-binding proteins and transporters, enzymes of flavonoid, jasmonate, polyamine, glutathione metabolism, and HSP70 proteins. Our data indicated the role of the phytohormone cytokinin in the observed responses. The metabolome profiling found that pipecolinic acid could be a part of the cadmium accumulation mechanism, and the observed accumulation of putrescine, coumaric acid, cinnamic acid, and coutaric acid confirmed the role of polyamines and flavonoids in tolerance to cadmium. In conclusion, our data provide new insight into cadmium tolerance and prospective targets for improving cadmium tolerance in other plants.

Cytokinin modulates the metabolic network of sulfur and glutathione.

The phytohormone cytokinin is implicated in a range of growth, developmental, and defense processes. A growing body of evidence supports a crosstalk between cytokinin and nutrient signaling pathways, such as nitrate availability. Cytokinin signaling regulates sulfur-responsive gene expression, but the underlying molecular mechanisms and their impact on sulfur-containing metabolites have not been systematically explored. Using a combination of genetic and pharmacological tools, we investigated the interplay between cytokinin signaling and sulfur homeostasis. Exogenous cytokinin triggered sulfur starvation-like gene expression accompanied by a decrease in sulfate and glutathione content. This process was uncoupled from the activity of the major transcriptional regulator of sulfate starvation signaling SULFUR LIMITATION 1 and an important glutathione-degrading enzyme, γ-glutamyl cyclotransferase 2;1, expression of which was robustly up-regulated by cytokinin. Conversely, glutathione accumulation was observed in mutants lacking the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 3 and in cytokinin-deficient plants. Cytokinin-deficient plants displayed improved root growth upon exposure to glutathione-depleting chemicals which was attributed to a higher capacity to maintain glutathione levels. These results shed new light on the interplay between cytokinin signaling and sulfur homeostasis. They position cytokinin as an important modulator of sulfur uptake, assimilation, and remobilization in plant defense against xenobiotics and root growth.

Type-A response regulators negatively mediate heat stress response by altering redox homeostasis in Arabidopsis.

Besides the long-standing role of cytokinins (CKs) as growth regulators, their current positioning at the interface of development and stress responses is coming into recognition. The current evidence suggests the notion that CKs are involved in heat stress response (HSR), however, the role of CK signaling components is still elusive. In this study, we have identified a role of the CK signaling components type-A Arabidopsis response regulators (ARRs) in HSR in Arabidopsis. The mutants of multiple type-A ARR genes exhibit improved basal and acquired thermotolerance and, altered response to oxidative stress in our physiological analyses. Through proteomics profiling, we show that the type-A arr mutants experience a 'stress-primed' state enabling them to respond more efficiently upon exposure to real stress stimuli. A substantial number of proteins that are involved in the heat-acclimatization process such as the proteins related to cellular redox status and heat shock, are already altered in the type-A arr mutants without a prior exposure to stress conditions. The metabolomics analyses further reveal that the mutants accumulate higher amounts of α-and γ-tocopherols, which are important antioxidants for protection against oxidative damage. Collectively, our results suggest that the type-A ARRs play an important role in heat stress response by affecting the redox homeostasis in Arabidopsis.

Cytokinin Deficiency Alters Leaf Proteome and Metabolome during Effector-Triggered Immunity in Arabidopsis thaliana Plants.

The involvement of cytokinins (CK) in biotic stresses has been recognized, while knowledge regarding the effects of CK deficiency on plant response against pathogens is less abundant. Thus, the purpose of this study was to reveal the effects of CK deficiency on proteomics and metabolomic responses of flg22-triggered immunity. We conducted a series of histochemical assays to investigate the activity of the downstream pathways caused by flg22, such as accumulation of ROS, induction of defence genes, and callose deposition, that occurred in Arabidopsis thaliana transgenic lines overexpressing the Hordeum vulgare CKX2 gene (HvCKX2), which are therefore CK-deficient. We also used GC and LC-MS-based technology to quantify variations in stress hormone levels and metabolomic and proteomic responses in flg22-treated HvCKX2 and wild-type Arabidopsis plants. We found that CK deficiency alters the flg22-triggered plant defence response, especially through induction of callose deposition, upregulation of defence response-related proteins, increased amino acid biosynthesis, and regulation of plant photosynthesis. We also indicated that JA might be an important contributor to immune response in plants deficient in CKs. The present study offers new evidence on the fundamental role of endogenous CK in the response to pathogens, as well as the possibility of altering plant biotic tolerance by manipulating CK pools.

Interaction With Fungi Promotes the Accumulation of Specific Defense Molecules in Orchid Tubers and May Increase the Value of Tubers for Biotechnological and Medicinal Applications: The Case Study of Interaction Between Dactylorhiza sp. and Tulasnella calospora.

Terrestrial orchids can form tubers, organs modified to store energy reserves. Tubers are an attractive source of nutrients, and salep, a flour made from dried orchid tubers, is the source of traditional beverages. Tubers also contain valuable secondary metabolites and are used in traditional medicine. The extensive harvest of wild orchids is endangering their populations in nature; however, orchids can be cultivated and tubers mass-produced. This work illustrates the importance of plant-fungus interaction in shaping the content of orchid tubers in vitro. Orchid plants of Dactylorhiza sp. grown in asymbiotic culture were inoculated with a fungal isolate from Tulasnella calospora group and, after 3 months of co-cultivation, tubers were analyzed. The fungus adopted the saprotrophic mode of life, but no visible differences in the morphology and biomass of the tubers were detected compared to the mock-treated plants. To elucidate the mechanisms protecting the tubers against fungal infestation, proteome, metabolome, and lipidome of tubers were analyzed. In total, 1,526, 174, and 108 proteins, metabolites, and lipids were quantified, respectively, providing a detailed snapshot of the molecular process underlying plant-microbe interaction. The observed changes at the molecular level showed that the tubers of inoculated plants accumulated significantly higher amounts of antifungal compounds, including phenolics, alkaloid Calystegine B2, and dihydrophenanthrenes. The promoted antimicrobial effects were validated by observing transient inhibition of Phytophthora cactorum growth. The integration of omics data highlighted the promotion of flavonoid biosynthesis, the increase in the formation of lipid droplets and associated production of oxylipins, and the accumulation of auxin in response to T. calospora. Taken together, these results provide the first insights into the molecular mechanisms of defense priming in orchid tubers and highlight the possible use of fungal interactors in biotechnology for the production of orchid secondary metabolites.

Differences in the Proteomic and Metabolomic Response of Quercus suber and Quercus variabilis During the Early Stages of Phytophthora cinnamomi Infection.

Phytophthora cinnamomi Rands is a cosmopolite pathogen of woody plants which during the last couple of centuries has spread all over the world from its center of origin in Southeast Asia. In contrast to Chinese cork oak (Quercus variabilis Blume) forests native to Asia, which are generally healthy despite the presence of the pathogen, the populations of Cork oaks (Quercus suber L.) in Europe have been severely decimated by P. cinnamomi. The present study aims at identifying the differences in the early proteomic and metabolomic response of these two tree species that lead to their differences in susceptibility to P. cinnamomi. By using micropropagated clonal plants, we tried to minimize the plant-to-plant differences in the defense response that is maximized by the high intraspecific genetic variability inherent to the Quercus genus. The evolution on the content of Phytophthora proteins in the roots during the first 36 h after inoculation suggests a slower infection process in Q. variabilis plants. These plants displayed a significant decrease in sugars in the roots, together with a downregulation of proteins related to carbon metabolism. In the leaves, the biggest changes in proteomic profiling were observed 16 h after inoculation, and included increased abundance of peroxidases, superoxide dismutases and glutathione S-transferases in Q. variabilis plants, which probably contributed to decrease its susceptibility to P. cinnamomi.

Light Quality Modulates Plant Cold Response and Freezing Tolerance.

The cold acclimation process is regulated by many factors like ambient temperature, day length, light intensity, or hormonal status. Experiments with plants grown under different light quality conditions indicate that the plant response to cold is also a light-quality-dependent process. Here, the role of light quality in the cold response was studied in 1-month-old Arabidopsis thaliana (Col-0) plants exposed for 1 week to 4°C at short-day conditions under white (100 and 20 µmol m-2s-1), blue, or red (20 µmol m-2s-1) light conditions. An upregulated expression of CBF1, inhibition of photosynthesis, and an increase in membrane damage showed that blue light enhanced the effect of low temperature. Interestingly, cold-treated plants under blue and red light showed only limited freezing tolerance compared to white light cold-treated plants. Next, the specificity of the light quality signal in cold response was evaluated in Arabidopsis accessions originating from different and contrasting latitudes. In all but one Arabidopsis accession, blue light increased the effect of cold on photosynthetic parameters and electrolyte leakage. This effect was not found for Ws-0, which lacks functional CRY2 protein, indicating its role in the cold response. Proteomics data confirmed significant differences between red and blue light-treated plants at low temperatures and showed that the cold response is highly accession-specific. In general, blue light increased mainly the cold-stress-related proteins and red light-induced higher expression of chloroplast-related proteins, which correlated with higher photosynthetic parameters in red light cold-treated plants. Altogether, our data suggest that light modulates two distinct mechanisms during the cold treatment - red light-driven cell function maintaining program and blue light-activated specific cold response. The importance of mutual complementarity of these mechanisms was demonstrated by significantly higher freezing tolerance of cold-treated plants under white light.

Transcriptome, metabolome and suppressor analysis reveal an essential role for the ubiquitin-proteasome system in seedling chloroplast development.

BACKGROUND: Many regulatory circuits in plants contain steps of targeted proteolysis, with the ubiquitin proteasome system (UPS) as the mediator of these proteolytic events. In order to decrease ubiquitin-dependent proteolysis, we inducibly expressed a ubiquitin variant with Arg at position 48 instead of Lys (ubK48R). This variant acts as an inhibitor of proteolysis via the UPS, and allowed us to uncover processes that are particularly sensitive to UPS perturbation. RESULTS: Expression of ubK48R during germination leads to seedling death. We analyzed the seedling transcriptome, proteome and metabolome 24 h post ubK48R induction and confirmed defects in chloroplast development. We found that mutations in single genes can suppress seedling lethality, indicating that a single process in seedlings is critically sensitive to decreased performance of the UPS. Suppressor mutations in phototropin 2 (PHOT2) suggest that a contribution of PHOT2 to chloroplast protection is compromised by proteolysis inhibition. CONCLUSIONS: Overall, the results reveal protein turnover as an integral part of a signal transduction chain that protects chloroplasts during development.

Regulation of heat shock proteins 70 and their role in plant immunity.

Heat shock proteins 70 (HSP70s) are steadily gaining more attention in the field of plant biotic interactions. Though their regulation and activity in plants are much less well characterized than are those of their counterparts in mammals, accumulating evidence indicates that the role of HSP70-mediated defense mechanisms in plant cells is indispensable. In this review, we summarize current knowledge of HSP70 post-translational control in plants. We comment on the phytohormonal regulation of HSP70 expression and protein abundance, and identify a prominent role for cytokinin in HSP70 control. We outline HSP70s' subcellular localizations, chaperone activity, and chaperone-mediated protein degradation. We focus on the role of HSP70s in plant pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity, and discuss the contribution of different HSP70 subfamilies to plant defense against pathogens.

Light Quality and Intensity Modulate Cold Acclimation in Arabidopsis.

Plant survival in temperate zones requires efficient cold acclimation, which is strongly affected by light and temperature signal crosstalk, which converge in modulation of hormonal responses. Cold under low light conditions affected Arabidopsis responses predominantly in apices, possibly because energy supplies were too limited for requirements of these meristematic tissues, despite a relatively high steady-state quantum yield. Comparing cold responses at optimal light intensity and low light, we found activation of similar defence mechanisms-apart from CBF1-3 and CRF3-4 pathways, also transient stimulation of cytokinin type-A response regulators, accompanied by fast transient increase of trans-zeatin in roots. Upregulated expression of components of strigolactone (and karrikin) signalling pathway indicated involvement of these phytohormones in cold responses. Impaired response of phyA, phyB, cry1 and cry2 mutants reflected participation of these photoreceptors in acquiring freezing tolerance (especially cryptochrome CRY1 at optimal light intensity and phytochrome PHYA at low light). Efficient cold acclimation at optimal light was associated with upregulation of trans-zeatin in leaves and roots, while at low light, cytokinin (except cis-zeatin) content remained diminished. Cold stresses induced elevation of jasmonic acid and salicylic acid (in roots). Low light at optimal conditions resulted in strong suppression of cytokinins, jasmonic and salicylic acid.

Split-root systems: detailed methodology, alternative applications, and implications at leaf proteome level.

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.

The Omics Hunt for Novel Molecular Markers of Resistance to Phytophthora infestans.

Wild Solanum accessions are a treasured source of resistance against pathogens, including oomycete Phytophthora infestans, causing late blight disease. Here, Solanum pinnatisectum, Solanum tuberosum, and the somatic hybrid between these two lines were analyzed, representing resistant, susceptible, and moderately resistant genotypes, respectively. Proteome and metabolome analyses showed that the infection had the highest impact on leaves of the resistant plant and indicated, among others, an extensive remodeling of the leaf lipidome. The lipidome profiling confirmed an accumulation of glycerolipids, a depletion in the total pool of glycerophospholipids, and showed considerable differences between the lipidome composition of resistant and susceptible genotypes. The analysis of putative resistance markers pinpointed more than 100 molecules that positively correlated with resistance including phenolics and cysteamine, a compound with known antimicrobial activity. Putative resistance protein markers were targeted in an additional 12 genotypes with contrasting resistance to P. infestans. At least 27 proteins showed a negative correlation with the susceptibility including HSP70-2, endochitinase B, WPP domain-containing protein, and cyclase 3. In summary, these findings provide insights into molecular mechanisms of resistance against P. infestans and present novel targets for selective breeding.

Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants.

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

Peptide-Based Identification of Phytophthora Isolates and Phytophthora Detection in Planta.

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.