Low), and also the ion chromatograms of all significant remaining ions (such as nonfragmented ones as well as fragment ions produced within the ESI supply) were extracted with a precision of .mz.From these, we selected significant ions showing big adjustments in peak areas in response to Fe deficiency, devoid of taking into consideration fragments and minor ions.The localization within the chromatograms from the chosen compounds is depicted in Figure , as well as the RT, exact mz and assigned elemental formulae are shown in Table .These compounds had been never ever detected in nutrient solutions of pots without having plants, and include some coumarins already recognized to take place and others not previously reported, as explained in detail beneath.Coumarins and Connected Compounds Previously Reported inside a.thaliana upon FeDeficiencyAs expected, some compounds (five out of) have RTs and mz values matching with these of coumarins previously discovered in roots and exudates from Fedeficient A.thaliana plants (Fourcroy et al Schmid et al Schmidt et al).These consist of compounds , , and (Figure ; Table), and were assigned to scopoletin hexoside, fraxetin, scopoletin, isofraxidin and fraxinol (an isofraxidin isomer), respectively (Supplementary Table S).These annotations had been additional confirmed applying the RT and mz values of requirements (Table vs.Table).A sixth compound, , was assigned to ferulic acid hexoside primarily based around the presence of a significant ion at mz .in its positive MS(TOF) spectrum, that is consistent together with the elemental formula of ferulic acid [MH] ion (Table) and together with the neutral loss of a hexosyl moiety (.Da, C H O) from the [MH] ion (with an absolute error of .ppm).We couldn’t confirm the SC66 Purity & Documentation identity using a ferulic acid hexoside common because towards the finest of our knowledge no such regular is commercially available.The remaining compounds have been subjected to additional MSbased analyses to get structural data.1st, low resolution HPLCESIMS(ion trap) analyses had been carried out, such as MS andor MS experiments using the [MH] or [MH] ions.Identification of Phenolic Compounds Induced by Fe Deficiency as Coumarins, Coumarin Precursors and CoumarinolignansTo recognize the compounds discovered in the A.thaliana root extracts and development media, samples had been analyzed employing four distinctive HPLCUVVISESIMS(TOF) protocols, which includes two Elution programs ( and ; Supplementary Table S) and two electrospray (ESI) ionization modes (constructive and damaging).The newly created Elution system led to a superior separation of phenolic compounds than that obtained with all the original Elution system used in Fourcroy et al..With all the new elution system, RTs to get a selected set of phenolics requirements ranged from .(for esculin, the glucoside form of the coumarin esculetin) to .min (for the flavone apigenin) (Supplementary Figures S and S).These HPLCESIMS(TOF) analyses supplied very accurate (error beneath ppm) measurements in the masstocharge (mz) ratio in the detected ions, hence enabling for correct elemental formulae assignments (Bristow,).Raw MS(TOF) datasets (time, mz and ion intensity) in the root extracts and nutrient options from Fedeficient and Fesufficient plants have been initial analyzed with all the DISSECT algorithm (Data Analysis PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21542721 .; Bruker) to obtain mass spectralCoumarins and CoumarinPrecursor Hexosides Not Previously Reported in Arabidopsis upon FeDeficiencyThree in the compounds (, and) had been identified as ferulic acid, coniferyl aldehyde and sinapyl aldehyde (3 phenylpropanoid precursors; Figure B), respectively, by comparing.
