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.

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.

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.

Metabolomic and Physiological Changes in Fagus sylvatica Seedlings Infected with Phytophthora plurivora and the A1 and A2 Mating Types of P. ×cambivora.

Phytophthora infections are followed by histological alterations, physiological and metabolomic adjustments in the host but very few studies contemplate these changes simultaneously. Fagus sylvatica seedlings were inoculated with A1 and A2 mating types of the heterothallic P. ×cambivora and with the homothallic P. plurivora to identify plant physiological and metabolomic changes accompanying microscope observations of the colonization process one, two and three weeks after inoculation. Phytophthora plurivora-infected plants died at a faster pace than those inoculated with P. ×cambivora and showed higher mortality than P. ×cambivora A1-infected plants. Phytophthora ×cambivora A1 and A2 caused similar progression and total rate of mortality. Most differences in the physiological parameters between inoculated and non-inoculated plants were detected two weeks after inoculation. Alterations in primary and secondary metabolites in roots and leaves were demonstrated for all the inoculated plants two and three weeks after inoculation. The results indicate that P. plurivora is more aggressive to Fagus sylvatica seedlings than both mating types of P. ×cambivora while P. ×cambivora A1 showed a slower infection mode than P. ×cambivora A2 and led to minor plant metabolomic adjustments.

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.

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.

Integrated Proteomic and Metabolomic Profiling of Phytophthora cinnamomi Attack on Sweet Chestnut (Castanea sativa) Reveals Distinct Molecular Reprogramming Proximal to the Infection Site and Away from It.

Phytophthora cinnamomi is one of the most invasive tree pathogens that devastates wild and cultivated forests. Due to its wide host range, knowledge of the infection process at the molecular level is lacking for most of its tree hosts. To expand the repertoire of studied Phytophthora-woody plant interactions and identify molecular mechanisms that can facilitate discovery of novel ways to control its spread and damaging effects, we focused on the interaction between P. cinnamomi and sweet chestnut (Castanea sativa), an economically important tree for the wood processing industry. By using a combination of proteomics, metabolomics, and targeted hormonal analysis, we mapped the effects of P. cinnamomi attack on stem tissues immediately bordering the infection site and away from it. P. cinnamomi led to a massive reprogramming of the chestnut proteome and accumulation of the stress-related hormones salicylic acid (SA) and jasmonic acid (JA), indicating that stem inoculation can be used as an easily accessible model system to identify novel molecular players in P. cinnamomi pathogenicity.

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.

Interplay between 1-aminocyclopropane-1-carboxylic acid, γ-aminobutyrate and D-glucose in the regulation of high nitrate-induced root growth inhibition in maize.

Nitrogen is one of the main factors that affect plant growth and development. However, high nitrogen concentrations can inhibit both shoot and root growth, even though the processes involved in this inhibition are still unknown. The aim of this work was to identify the metabolic alterations that induce the inhibition of root growth caused by high nitrate supply, when the whole plant growth is also reduced. High nitrate altered nitrogen and carbon metabolism, reducing the content of sugars and inducing the accumulation of Ca2+ and amino acids, such as glutamate, alanine and γ-aminobutyrate (GABA), that could act to replenish the succinate pool in the tricarboxylic acid cycle and maintain its activity. Other metabolic alterations found were the accumulation of the polyamines spermidine and spermine, and the reduction of jasmonic acid (JA) and the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC). These results indicate that the growth root inhibition by high NO3- is a complex metabolic response that involves GABA as a key link between C and N metabolism which, together with plant growth regulators such as auxins, cytokinins, abscisic acid, JA, and the ethylene precursor ACC, is able to regulate the metabolic response of root grown under high nitrate concentrations.

Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.

The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves.

The imbalance between C and N metabolism during high nitrate supply inhibits photosynthesis and overall growth in maize (Zea mays L.).

Nitrogen (N) is an important regulator of photosynthetic carbon (C) flow in plants, and an adequate balance between N and C metabolism is needed for correct plant development. However, an excessive N supply can alter this balance and cause changes in specific organic compounds associated with primary and secondary metabolism, including plant growth regulators. In previous work, we observed that high nitrate supply (15 mM) to maize plants led to a decrease in leaf expansion and overall biomass production, when compared with low nitrate supply (5 mM). Thus, the aim of this work is to study how overdoses of nitrate can affect photosynthesis and plant development. The results show that high nitrate doses greatly increased amino acid production, which led to a decrease in the concentration of 2-oxoglutarate, the main source of C skeletons for N assimilation. The concentration of 1-aminocyclopropane-1-carboxylic acid (and possibly its product, ethylene) also rose in high nitrate plants, leading to a decrease in leaf expansion, reducing the demand for photoassimilates by the growing tissues and causing the accumulation of sugars in source leaves. This accumulation of sugars, together with the decrease in 2-oxoglutarate levels and the reduction in chlorophyll concentration, decreased plant photosynthetic rates. This work provides new insights into how high nitrate concentration alters the balance between C and N metabolism, reducing photosynthetic rates and disrupting whole plant development. These findings are particularly relevant since negative effects of nitrate in contexts other than root growth have rarely been studied.

Plant responses to ambient temperature fluctuations and water-limiting conditions: A proteome-wide perspective.

BACKGROUND: Every year, environmental stresses such as limited water and nutrient availability, salinity, and temperature fluctuations inflict significant losses on crop yields across the globe. Recently, developments in analytical techniques, e.g. mass spectrometry, have led to great advances towards understanding how plants respond to environmental stresses. These processes are mediated by many molecular pathways and, at least partially, via proteome-environment interactions. SCOPE OF REVIEW: This review focuses on the current state of knowledge about interactions between the plant proteome and the environment, with a special focus on drought and temperature responses of plant proteome dynamics, and subcellular and organ-specific compartmentalization, in Arabidopsis thaliana and crop species. MAJOR CONCLUSIONS: Correct plant development under non-optimal conditions requires complex self-protection mechanisms, many of them common to different abiotic stresses. Proteome analyses of plant responses to temperature and drought stresses have revealed an intriguing interplay of modifications, mainly affecting the photosynthetic machinery, carbohydrate metabolism, and ROS activation and scavenging. Imbalances between transcript-level and protein-level regulation observed during adaptation to abiotic stresses suggest that many of the regulatory processes are controlled at translational and post-translational levels; proteomics is thus essential in revealing important regulatory networks. GENERAL SIGNIFICANCE: Because information from proteomic data extends far beyond what can be deduced from transcriptome analysis, the results of proteome studies have substantially deepened our understanding of stress adaptation in plants; this is clearly a prerequisite for designing strategies to improve the yield and quality of crops grown under unfavorable conditions brought about by ongoing climatic change. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

High nitrate supply reduces growth in maize, from cell to whole plant.

Nitrogen (N) is an essential macronutrient that limits agricultural productivity, and both low and high N supply have been suggested to alter plant growth. The overall aim of this work is to study the impact of nitrate (NO3(-)) in maize yield and the possible causes that induce this alteration. High NO3(-) doses did not increase the yield of maize grown neither in the field nor under controlled conditions. In fact, plants grown under controlled conditions for 45 days with NO3(-) concentrations over 5mM showed a decrease in biomass production. This reduction was perceptible in shoots prior to roots, where phytomer expansion was reduced. Cell size and number were also reduced in the leaves of plants with high NO3(-). This alteration was correlated with the increase of 1-aminocyclopropane-1-carboxylic acid in leaves, which was probably translocated from the roots in order to synthesize ethylene. Cytokinins (CKs) also showed a relevant role in this inhibitory effect, increasing in high NO3(-) plants with a reduction in root and shoot growth, inhibition of apical dominance and a strong decrease of leaf expansion, symptoms described previously as "CK syndrome". We propose that high NO3(-) inhibits maize growth by causing hormonal alterations that modify plant growth from cell to whole plant.