After the fusion of Tic20-proteoliposomes with a lipid bilayer, ion channel activity was observed . The total conductance under symmetrical buffer conditions , 250 mM KCl was dependent on the direction of the applied potential: 1260 pS and 1010 pS under negative and positive voltage values, respectively. The channel was mostly in the completely open state, however, individual single gating events were also frequently observed, varying in a broad range between 25 pS to 600 pS . All detected gating events were depicted in two histograms . Two conductance classes were defined both at negative and positive voltage values with thresholds of 220 pS and 180 pS, respectively . Note that gating events belonging to the smaller conductance classes occurred more frequently. The observed pore seems to be asymmetric, since higher conductance classes notably differ under positive and negative voltages. This is probably due to interactions of the permeating ions with the channel, which presumably exhibits an asymmetric potential profile along the pore. Since small and large opening events were simultaneously observed in all experiments, it is very unlikely that they belong to two different pores. The selectivity of Tic20 was investigated under asymmetric salt conditions , 250/20 mM KCl. Similarly to the conductance values, the channel is intrinsically rectifying ,supporting asymmetric channel properties. The obtained reverse potential is 37.0 ± 1.4 mV . According to the Goldman-Hodgkin-Katz approach, this corresponds to a selectivity of 6.5:1 for K+ :Cl- -ions, thus indicating cation selectivity similar to Tic110. To determine the channel’s orientation within the bilayer, two side-specific characteristics were taken into account: the highest total conductance under symmetrical buffer conditions was measured under negative voltage values, and the channel rectifies in the same direction under asymmetrical buffer conditions . Therefore, it seems that the protein is randomly inserted into the bilayer. The pore size was roughly estimated according to Hille et al.. Considering the highest conductance class , a channel length of 1-5 nm and a resistivity of 247.5 Ω cm for a solution containing 250 mM KCl,frambueso maceta taking into account that the conductivity of the electrolyte solution within the pore is ~5 times lower than in the bulk solution, the pore size was estimated to vary between 7.8-14.1 Å.
This is in good agreement with the size of protein translocation channels such as Toc75 in the outer envelope membrane and Tic110 in the IE. Thus, the size of the Tic20 pore would be sufficient for the translocation of precursor proteins through the membrane. NtTic110, as a negative control, did not show any channel activity during electrophysiological measurements, indicating that the measured channel is not the result of a possible bacterial contamination . Considering our data presented here and those published in previous studies, we can conclude that the Tic translocon consists of distinct translocation channels: On the one hand, Tic110 forms the main translocation pore and therefore facilitates import of most of the chloroplast-targeted preproteins; on the other hand, Tic20 might facilitate the translocation of a subset of proteins. This scenario would match the one found in the inner mitochondrial membrane, where specific translocases exist for defined groups of precursor proteins: the import pathway of mitochondrial carrier proteins being clearly separated from that of matrix targeted preproteins. The situation in chloroplasts does not seem as clear-cut, but an analogous separation determined by the final destination and/or intrinsic properties of translocated proteins is feasible. The severe phenotype of attic20-I mutants prompts us to hypothesize that Tic20 might be specifically required for the translocation of some essential proteins. According to cross-linking results, Tic20 is connected to Toc translocon components. Therefore, after entering the intermembrane space via the Toc complex, some preproteins might be transported through the IE via Tic20. On the contrary, Kikuchi et al.presented that Tic20 migrates on BN-PAGE at the same molecular weight as the imported precursor of the small subunit of Rubisco and that tic20-I mutants display a reduced rate of the artificial precursor protein RbcS-nt: GFP. The authors interpreted these results in a way that Tic20 might function at an intermediate step between the Toc translocon and the channel of Tic110. However, being a substantial part of the general import pathway seems unlikely due to the very low abundance of Tic20. It is feasible to speculate that such abundant proteins as pSSU, which are imported at a very high rate, may interact incidentally with nearby proteins or indifferently use all available import channels.
To clarify this question, substrate proteins and interaction partners of Tic20 should be a matter of further investigation. Additionally, a very recent study suggested AtTic20-IV as an import channel working side by side with AtTic20-I. However, detailed characterization of the protein and experimental evidence for channel activity are still missing.Plants have pre-formed and inducible structural and biochemical mechanisms to prevent or arrest pathogen ingress and colonization. These defenses include barriers such as papillae and ligno-suberized layers to fortify cell walls, and low-molecular weight inhibitory chemicals . Plants undergo transcriptional changes upon perception of microbe associated molecular patterns or effectors to induce local and systemic resistance. The oomycete MAMPs, arachidonic acid and eicosapentaenoic acid , are potent elicitors of defense. These eicosapolyenoic acids were first identified as active components in Phytophthora infestans spore and mycelial extracts capable of eliciting a hypersensitive-like response, phytoalexin accumulation, lignin deposition, and protection against subsequent infection in potato tuber discs . Further work demonstrated root treatment with AA protects tomato and pepper seedlings from root and crown rot caused by Phytophthora capsici, with associated lignification at sites of attempted infection . AA has been shown to induce resistance, elicit production of reactive oxygen species, and trigger programmed cell death in members of the Solanaceae and other families . Phaeophyta and Rhodophyta members contain numerous bioactive chemicals that can elicit defense responses in plants . The brown alga, Ascophyllum nodosum, is a rich source of polyunsaturated fatty acids, including AA and EPA, which comprise nearly 25% of its total fatty acid composition . A. nodosum and oomycetes belong to the major eukaryotic lineage, the Stramenopila, and share other biochemical features . Commercial extracts of A. nodosum, used in organic and conventional agriculture as plant bio-stimulants, may also help plants cope with biotic and abiotic stresses. A proprietary A. nodosum extract, Acadian , has been shown to provide protection against bacterial and fungal pathogens . Studies in A. thaliana showed ANE induced systemic resistance to Pseudomonas syringae pv. tomato and Sclerotinia sclerotiorum . Investigation into ANE-induced resistance in A. thaliana and tomato suggest the role of ROS production, jasmonic acid signaling, and upregulation of defense-related genes and metabolites .
As a predominant polyunsaturated fatty acid in ANE, AA may contribute to ANE’s biological activity. In a parallel study we demonstrated AA’s ability to systemically induce resistance and ANE’s capacity to locally and systemically induce resistance in tomato to different pathogens . Further, we showed that AA and ANE altered the phytohormone profile of tomato by modulating the accumulation of defense-related phytohormones . Through RNA sequencing,cultivar frambuesas this same study revealed a striking level of transcriptional overlap in the gene expression profiles of AA- or ANEroot-treated tomato across tested time points . Gene ontology functional analysis of transcriptomic data revealed AA and ANE enriched similar categories of genes with nearly perfect overlap also observed in categories of under-represented genes. Both AA and ANE treatment protected seedings from challenge with pathogens with different parasitic strategies while eliciting expression of genes involved in immunity and secondary metabolism. The shared induced resistance phenotype and extensive transcriptional overlap of AA and ANE treatments suggested similar metabolic changes may be occurring in treated plants. In the current study, untargeted metabolomic analyses were conducted to assess global effects of root treatment with AA and the AA-containing complex extract, ANE, on the metabolome of tomato plants. Fatty acid sodium salts were prepared and stored as previously described . AA stock solution was prepared by dissolving 100 mg of fatty acid salt in 1 mL of 75% ethanol. AA stock solution was subsequently stored in a glass vial at −20°C flushed with N2 to minimize oxidation. A proprietary formulation of A. nodosum extract was diluted with deionized water to a 10% working concentration, which was used to prepare treatment dilutions. All chemicals were diluted to their treatment concentrations with sterile diH2O. Hydroponically reared, 3-weekold tomato seedlings with two fully expanded true leaves were transferred to 1 L darkened treatment containers with their respective root treatment solutions. Following 24, 48, 72, and 96 hours of root treatment, tomato seedlings were removed from treatment containers, and leaves and roots were excised from shoots and flash frozen in liquid nitrogen. Each sample was the pool of roots or leaves of two seedlings with four replications per tissue, treatment, and time point. Samples were transported on dry ice and stored at −70 °C until metabolite extraction. The issue samples were ground in liquid nitrogen using a mortar and pestle and 100 mg was weighed and transferred to a 2-ml bead-beating tube containing four 2.8-mm ceramic beads. All tools and consumables were pre-chilled in liquid nitrogen. After weighing, each sample was removed from liquid nitrogen and kept at −20 °C until addition of extraction solution.One ml of extraction solution was added to each sample which was then vortexed, followed by bead-beating in a bead mill at a speed of 2.9 m/s for one 3-min cycle. After bead-beating, samples were centrifuged at 12k × g for 10 min at 4 °C . Samples were diluted 5-fold using extraction solution and filtered into LC-MS-grade HPLC vials using 0.22-μm PTFE syringe filters. HPLC vials were kept at 4 °C until LC-MS analysis. A blank was prepared by adding 1 ml extraction solution to a bead-beating tube containing beads that was processed equivalently to the samples. In addition, a quality control sample was prepared by combining 20 μl of each of the extracted samples and processed equivalently.Samples were analyzed via high performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry controlled by MassHunter software in centroid data mode. Mobile phase A was ultrapure water with 0.05 % formic acid and mobile phase B was acetonitrile with 0.05 % formic acid. Before starting the run, the column , equipped with a guard column , was conditioned for 20 minutes with 95 % mobile phase A and 5 % B. Column temperature was maintained at 40 °C.
The sample injection order was randomized, with individual samples being run consecutively in positive and negative mode. The quality control sample was injected at the beginning and end of the run, as well as after every 12 samples throughout the run to check signal and elution stability. Source parameters were as follows: drying gas temperature of 325 °C and 350 °C , drying gas flow 12 l/min, nebulizer pressure 35 psi, sheath gas temp 375 °C and 400 °C , sheath gas flow 11 l/min, capillary voltage 3500 V and 3000 V , nozzle voltage 0 V and 1500 V , fragmentor 125 V, skimmer 65 V, and octopole 750 V. Acquisition was performed over a mass range of 50 to 1700 m/z using the all-ions MS/MS technique, cycling three different collision energies at an acquisition rate of 3 spectra/s. Simultaneous infusion of a solution of purine and hexakisphosphazine using the reference nebulizer was used throughout the runs for mass calibration. Positive and negative mode raw data files from MassHunter were analysed separately in MS-DIAL before downstream analysis. Tolerances for MS1 and MS2 were set to 0.025 and 0.075 Da respectively . For peak detection, the mass slice width was set to 0.1 DA and the minimum peak height was set to 15,000 which was approximately 3 times the noise level observed in the total ion chromatogram. A linear weighted moving average method was used for peak smoothing, with a smoothing level of 3 scans and a minimum peak width of 5 scans. Deconvolution was performed with a sigma window value of 0.5 and an MS/MS abundance cutoff of 10. The adducts permitted were [M+H]+, [M+NH4]+, [M+Na] +, [M+K]+, [M+H−H2O]+, and [2M+H]+ in positive mode, and [M−H]−, [M−H2O−H]−, [M+Cl]−, [M+Na−2H]−, and [M+K−2H]− in negative mode.MS-DIAL data was cleaned in MS-CleanR in RStudio using the following parameters: minimum blank ratio of 0.8, maximum relative standard deviation of 30, minimum relative mass deffect of 50, maximum RMD of 3000, maximum mass difference of 0.05 and maximum retention time difference of 0.15.