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 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.

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.

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.

Arabidopsis Response to Inhibitor of Cytokinin Degradation INCYDE: Modulations of Cytokinin Signaling and Plant Proteome.

Cytokinins are multifaceted plant hormones that play crucial roles in plant interactions with the environment. Modulations in cytokinin metabolism and signaling have been successfully used for elevating plant tolerance to biotic and abiotic stressors. Here, we analyzed Arabidopsis thaliana response to INhibitor of CYtokinin DEgradation (INCYDE), a potent inhibitor of cytokinin dehydrogenase. We found that at low nanomolar concentration, the effect of INCYCE on seedling growth and development was not significantly different from that of trans-Zeatin treatment. However, an alteration in the spatial distribution of cytokinin signaling was found at low micromolar concentrations, and proteomics analysis revealed a significant impact on the molecular level. An in-depth proteome analysis of an early (24 h) response and a dose-dependent response after 168 h highlighted the effects on primary and secondary metabolism, including alterations in ribosomal subunits, RNA metabolism, modulations of proteins associated with chromatin, and the flavonoid and phenylpropanoid biosynthetic pathway. The observed attenuation in stress-response mechanisms, including abscisic acid signaling and the metabolism of jasmonates, could explain previously reported positive effects of INCYDE under mild stress conditions.

Barley Root Proteome and Metabolome in Response to Cytokinin and Abiotic Stimuli.

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.

Eggplant Germination is Promoted by Hydrogen Peroxide and Temperature in an Independent but Overlapping Manner.

Hydrogen peroxide promotes seed germination, but the molecular mechanisms underlying this process are unclear. This study presents the results of eggplant (Solanum melongena) germination analyses conducted at two different temperatures and follows the effect of hydrogen peroxide treatment on seed germination and the seed proteome. Hydrogen peroxide was found to promote eggplant germination in a way not dissimilar to that of increased temperature stimuli. LC-MS profiling detected 729 protein families, 77 of which responded to a temperature increase or hydrogen peroxide treatment. These differentially abundant proteins were found to be involved in a number of processes, including protein and amino acid metabolism, carbohydrate metabolism, and the glyoxylate cycle. There was a very low overlap between hydrogen peroxide and temperature-responsive proteins, highlighting the differences behind the seemingly similar outcomes. Furthermore, the observed changes from the seed proteome indicate that hydrogen peroxide treatment diminished the seed endogenous hydrogen peroxide pool and that a part of manifested positive hydrogen peroxide effect might be related to altered sensitivity to abscisic acid.

Mechanistic insights into the evolution of DUF26-containing proteins in land plants.

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation. However, gene and genome duplications pose difficulties for functional characterization and translational research. Here we infer the evolutionary history of the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 emerged in secreted proteins. Domain duplications and rearrangements led to the appearance of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). The DUF26 is land plant-specific but structural analyses of PDLP ectodomains revealed strong similarity to fungal lectins and thus may constitute a group of plant carbohydrate-binding proteins. CRKs expanded through tandem duplications and preferential retention of duplicates following whole genome duplications, whereas PDLPs evolved according to the dosage balance hypothesis. We propose that new gene families mainly expand through small-scale duplications, while fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.

Affinity chromatography revealed 14-3-3 interactome of tomato (Solanum lycopersicum L.) during blue light-induced de-etiolation.

De-etiolation is the first developmental process under light control allowing the heterotrophic seedling to become autotrophic. The phytohormones cytokinins (CKs) largely contribute to this process. Reversible phosphorylation is a key event of cell signaling, allowing proteins to become active or generating a binding site for specific protein interaction. 14-3-3 proteins regulate a variety of plant responses. The expression, hormonal regulation, and proteomic network under the control of 14-3-3s were addressed in tomato (Solanum lycopersicum L.) during blue light-induced photomorphogenesis. Two isoforms were specifically investigated due to their high expression during tomato de-etiolation. The multidisciplinary approach demonstrated that TFT9 expression, but not TFT6, was regulated by CKs and identified cis-regulating elements required for this response. Our study revealed >130 potential TFT6/9 interactors. Their functional annotation predicted that TFTs might regulate the activity of proteins involved notably in cell wall strengthening or primary metabolism. Several potential interactors were also predicted to be CK-responsive. For the first time, the 14-3-3 interactome linked to de-etiolation was investigated and evidenced that 14-3-3s might be involved in CK signaling pathway, cell expansion inhibition and steady-state growth rate establishment, and reprograming from heterotrophy to autotrophy. BIOLOGICAL SIGNIFICANCE: Tomato (Solanum lycopersicum L.) is one of the most important vegetables consumed all around the world and represents probably the most preferred garden crop. Regulation of hypocotyl growth by light plays an important role in the early development of a seedling, and consequently the homogeneity of the culture. The present study focuses on the importance of tomato 14-3-3/TFT proteins in this process. We provide here the first report of 14-3-3 interactome in the regulation of light-induced de-etiolation and subsequent photomorphogenesis. Our data provide new insights into light-induced de-etiolation and open new horizons for dissecting the post-transcriptional regulations.

Hydrogen Peroxide: Its Role in Plant Biology and Crosstalk with Signalling Networks.

Hydrogen peroxide (H2O2) is steadily gaining more attention in the field of molecular biology research. It is a major REDOX (reduction⁻oxidation reaction) metabolite and at high concentrations induces oxidative damage to biomolecules, which can culminate in cell death. However, at concentrations in the low nanomolar range, H2O2 acts as a signalling molecule and in many aspects, resembles phytohormones. Though its signalling network in plants is much less well characterized than are those of its counterparts in yeast or mammals, accumulating evidence indicates that the role of H2O2-mediated signalling in plant cells is possibly even more indispensable. In this review, we summarize hydrogen peroxide metabolism in plants, the sources and sinks of this compound and its transport via peroxiporins. We outline H2O2 perception, its direct and indirect effects and known targets in the transcriptional machinery. We focus on the role of H2O2 in plant growth and development and discuss the crosstalk between it and phytohormones. In addition to a literature review, we performed a meta-analysis of available transcriptomics data which provided further evidence for crosstalk between H2O2 and light, nutrient signalling, temperature stress, drought stress and hormonal pathways.

Cytokinin at the Crossroads of Abiotic Stress Signalling Pathways.

Cytokinin is a multifaceted plant hormone that plays major roles not only in diverse plant growth and development processes, but also stress responses. We summarize knowledge of the roles of its metabolism, transport, and signalling in responses to changes in levels of both macronutrients (nitrogen, phosphorus, potassium, sulphur) and micronutrients (boron, iron, silicon, selenium). We comment on cytokinin's effects on plants' xenobiotic resistance, and its interactions with light, temperature, drought, and salinity signals. Further, we have compiled a list of abiotic stress-related genes and demonstrate that their expression patterns overlap with those of cytokinin metabolism and signalling genes.