A mix of accumulation and suppression of terpenoids and an increase of methoxyphenols was observed in the leaves of ANE-root-treated plants. Chemical enrichment analyses broadly revealed classes of metabolites that were induced or suppressed in AA- or ANE-treated plants. To examine which specific variables provide the strongest discriminatory power between the two treatment groups, a two-group comparative supervised multivariate analysis, orthogonal projections to latent structures discriminant analysis , was utilized. OPLS-DA score plots show strong between group variability discrimination between AA and ANE treatment groups compared to the H2O control across all tested time points with the x-axis describing the inter-treatment variability, and the y-axis showing the intra-treatment variability . S-plots derived from OPLS-DA were examined for both AA and ANE treatments in pairwise comparison with H2O control. S-plots of OPLS-DA revealed that treatment with AA or ANE induced shared changes in the levels of several defense-related metabolites in roots . Variables with the most negative and positive correlation and covariance values are the most influential in the model. These metabolites are located on either tail of the S-plot and contribute most greatly to the separation between treatment groups Bar charts depicting mean LC-MS signals for top OPLS-DA S-plot metabolites visualized across all time points illustrate that AA and ANE have similar effects on plant metabolic response .
Treatment of tomato roots with AA and ANE resulted in a sharp increase in metabolic intermediates in ligno-suberin biosynthesis. This includes AA-induced accumulation of moupinamide and significant increases in coniferyl alcohol in the roots of ANE-treated plants across all tested time points. In roots, AA and ANE treatments also induced increased levels of N-ethyl phydroxycinnamide and N1-trans-feruloylagmatine compared to H2O treatment,wholesale plant containers reflecting strong upregulation of the shikimate pathway and phenolic compound synthesis. Reduced levels of tomatine and dehydrotomatine were observed in the roots of AA- and ANE-treated plants indicating suppression of steroid glycoalkaloid biosynthesis. Treated plants also showed lower levels of lyso-phosphatidyl ethanolamine that could reflect enhanced membrane lipid turnover. AA and ANE can induce disease resistance locally and systemically, alter the accumulation of key phytohormones, and change the transcriptional profile of tomato with a striking level of overlap between the two treatments . The current study examined and characterized the AA- and ANE-induced metabolomes of tomato. AA and ANE locally and systemically induce metabolome remodeling toward defense-associated metabolic features. Early studies investigating transcriptional and metabolic changes in potato revealed selective partitioning and shifting of terpenoid biosynthesis from steroidal glycoalkaloids to sesquiterpenes following treatment with AA or EPA or challenged with P. infestans . Similarly, our work here with AA- and ANE-treated tomato seedlings has shown a marked decrease in the levels of two abundant glycoalkaloids, tomatine and dehydrotomatine .
Our data also show strong enrichment of sesquiterpenes in leaves of AA-treated plants at 96 hours post treatment, although the identity of these sesquiterpenes is unresolved . This work further supports evidence for differential regulation and sub-functionalization of sterol/ glycoalkaloid and sesquiterpene biosynthetic pathways in solanaceous plants in different stress contexts . AA and EPA are strong elicitors that are abundant in structural and storage lipids of oomycete pathogens, but absent from higher plants. Although their initial perception by the plant is likely different from that of canonical MAMPs , there is some convergence in downstream defenses induced by these various MAMPs. Work to characterize the effect of canonical MAMP treatment on the metabolomes of various plant species has implicated common metabolic changes that prime for defense. Cells and leaf tissue of A. thaliana treated with lipopolysaccharide showed enrichment of phenylpropanoid pathway metabolites, including cinnamic acid derivatives and glycosides . In the same study, SA and JA were also positively correlated with LPS treatment, as we also observed in tomato following treatment with AA . Recent work in A. thaliana wild-type and receptor mutants treated with two chemotypes of LPS showed increases in hydroxycinnamic acid and derivatives and enrichment of the associated phenylpropanoid pathway . Work in tobacco similarly found treatment with LPS, chitosan, and flg22 all induced accumulation of hydroxycinnamic acid and derivatives, and that defense responses elicited by these MAMPs were modulated by both SA and JA . More recent work in the cells of Sorghum bicolor treated with LPS showed enrichment of hydroxycinnamic acids and other phenylpropanoids in coordination with accumulation of both SA and JA . Treatment of tomato with flg22 and flgII-28 also enriched hydroxycinnamic acids, and tomato treatment with cps22 revealed a metabolic shift toward the phenylpropanoid pathway with hydroxycinnamic acid, conjugates and derivatives as key biomarkers .
Similar to traditional MAMPs, AA and the AA/EPA-containing complex mixture, ANE, both induce enrichment of cinnamic acid and derivatives in tomato seedlings . This supports the hypothesis that MAMPs broadly induce similar metabolic changes to enrich pools of specialized secondary metabolites that contribute to plant immunity. AA- and ANE-treated roots showed strong enrichment of metabolic features classified as fatty acyl glycosides of mono- and disaccharides . Fatty acyl glycosides have been studied in several plant families and are most extensively characterized in members of Solanaceae . Investigations into the function of fatty acyl glycosides in plants suggest they may act to protect against insect herbivory through various mechanisms and provide protection against fungal pathogens . A recent study isolated and identified fatty acyl glycosides from strawberry capable of inducing immune responses in A. thaliana, including ROS burst, callose deposition, increased expression of defense-related genes, and induced resistance to bacterial and fungal challenge . This same work also demonstrated that the strawberry-derived fatty acyl glycosides induced resistance in soybean and, due to their antimicrobial activity, also protected lemon fruits post harvest from fungal infection . AA- and ANE-root treatments locally elicit accumulation of the same class of defense associated metabolites that Grellet et al. illustrated to have direct antimicrobial activity and protect against disease . Cell wall fortification is an important plant defense often initiated upon pathogen infection. Cell wall lignification is a well-studied mechanism with localized accumulation of phenolic intermediates and lignin at attempted penetration sites . Lignification reinforces and rigidifies the cell wall to create an impervious barrier to microbial ingress . In our study, AA treatment of tomato roots induced accumulation of a phenylcoumaran intermediate in lignin biosynthesis, while ANE treatment induced accumulation of coniferyl alcohol, an important monomer unit of lignin. Interestingly, coniferyl alcohol has recently been shown to act in a signaling capacity in a regulatory feedback mechanism to intricately control lignin biosynthesis, an irreversible process that is energetically costly . The findings of our study coincide with the well-characterized role of lignin and its intermediates in plant defense. This work characterizes local and systemic metabolic profiles of AA- and ANE-treated tomato with the oomycete-derived MAMP, AA,plastic pot manufacturers and the AA-containing biostimulant, ANE. AA and ANE profoundly alter the tomato metabolome toward defense-associated secondary metabolites with notable overlap in enriched metabolite classes compared to H2O control. Further investigation is required to elucidate the functional contribution of these metabolic features in AA- and ANE induced resistance and, more broadly, plant immunity. Our studyadds to the understanding of MAMP-induced metabolomes with implications for further development of seaweed-derived biostimulants for crop improvement.Although the major N forms available to crop plants in most agricultural soils are ammonium and nitrate, urea is found ubiquitously in the soil and additionally represents the most widespread form of nitrogen fertilizer used in agricultural plant production. Due to its fast hydrolysis by urease the concentration of urea in agricultural soils is in general below 70 µM and most of the urea derived nitrogen is thought to be taken up from the soil solution in the form of ammonium .
However, biphasic uptake kinetics of labelled urea in Chara cells indicated that membrane transport systems might facilitate the uptake of urea into plant cells . By heterologous expression of plant genes in yeast and frog oocytes, two different transport systems were identified, that allow the membrane permeation of urea. Several aquaporins of the TIP, NIP and PIP families have been identified as low affinity transport systems , while AtDUR3 encodes a H+ /urea cotransporter , that facilitates high affinity urea transport in heterologous systems. In addition to externally supplied urea, internal production of urea represents an important intermediate of the N metabolism in higher plants. Urea is amongst others increasingly generated during protein degradation, a process of particular importance for the retranslocation of nitrogen during plant senescence. In senescence the earliest and most drastic change in plant cellular structures is the breakdown of chloroplasts, which hold the majority of the leaf protein . The degradation of these photosynthetic proteins results in the generation of mostly free amino acids, in particular arginine. This is assimilated in the mitochondria within the ornithine cycle by degradation into ornithine and urea . Urease is thereby the only enzyme known in plants that is able to recapture nitrogen from urea, which otherwise would remain an unavailable N source. It has been localized to the cytoplasm and was detected in particular in generative tissues of plants , although it seems to be synthesized in almost all organs . The product of the urease reaction, ammonia, is then assimilated via the GS-GOGAT pathway into glutamine . To date it is unclear if urea also has a meaning for short-term storage and long-distance transport of N. Cadmium is one of the most widespread hazardous metals found in agricultural soils. In a market-basket study conducted in Japan, 7% of 381 eggplant samples were found to exceed the maximum Cd concentration for fruiting vegetables recommended by codex Alimentarius Commissions. These results indicate the urgent requirement for cultivation systems that reduce the Cd concentration in eggplant. Grafting eggplant Solanum melongena onto Solanum torvum has been reported to be an effective method for reducing the Cd concentration in the fruits of eggplant by up to one-fourth.1). The Cd concentration in the shoot and xylem sap of S. melongena was higher than that of S. torvum; however, the concentration in the roots of both species was almost identical.2). It was likely that the limited translocation of Cd into the shoot of S. torvum was partly due to the presence of a barrier function in root tissues. In this study, we compared the microscale distribution pattern of Cd in root tissues of S. melongena and S. torvum by using the synchrotron micro-X-ray fluorescence technique and thus elucidated the role of the roots of S. torvum in restricting Cd transport to the shoot. Hydroponically cultivated S. melongena cv. Senryo 2 and S. torvum cv. Torubamubiga were exposed to CdCl2 for 24 h with fresh nutrient solution at pH 5.5 in a controlled environment chamber. From 5-mm root segments obtained at a distance of 10 and 40 mm from the root apex, 200-μm thick transverse sections were prepared using a microtome. The freshly prepared sections were frozen immediately by contacting them on an aluminum block with a mirrored surface that was pre-cooled by liquid nitrogen; the sections were subsequently freeze-dried. The freeze-dried sections were then mounted on an acrylic plate for SR m-XRF analysis. A few of the sections were stained with berberine hemisulfate/aniline blue.3) to identify the presence of Casparian bands by fluorescence microscopy. The distribution of Cd, Zn, and Fe in the transverse sections of the root was mapped using SR -XRF at BL37XU, SPring-8, Japan Synchrotron Radiation Research Institute. The beam spot size was 2.0 m wide and 1.0 m in height, and the scanning step size was 3 m. The spectral acquisition time was set to 2 s. Although copper-based engineered nanomaterials currently comprise a relatively small fraction of global ENM production ,1 their toxicity and life cycle characteristics raise concerns regarding their environmental risk. For example, a common use for Cu-based ENMs is as the active ingredient in marine antifouling paints or agricultural biocides,2 where they are directly introduced into the environment as intentionally toxic substances. Copper based ENMs are somewhat unique among the most widely used ENMs in that they can participate in redox reactions to form three oxidation states: Cu0 , Cu1+, and Cu2+. Copper can also participate in a number of inorganic complexes with compounds found in natural waters, such as sulfate, sulfide, phosphate, chloride, and carbonate.