Deficiency remedy, straight away frozen in liquid N , and stored at C for RNA extraction.Nutrient options were sampled at days and just after the onset of Fe deficiency remedy, and straight away stored at C until extraction of phenolic compounds.Shoots and roots have been sampled separately in the finish in the experimental period.Leaf disks (.cm .cm) have been taken from young leaves and stored at C for photosynthetic pigment analysis.Roots had been washed with tap water then with sort I water,Mineral AnalysisPlant tissues have been ground and digested as indicated in Fourcroy et al..Iron, Mn, Cu, and Zn had been determined by flame atomic absorption spectrometry employing a SOLAAR (+)-Viroallosecurinine Epigenetic Reader Domain apparatus (Thermo, Cambridge, UK).Extraction of Phenolic Compounds from Roots and Nutrient SolutionsPhenolic compounds were extracted from roots and nutrient options as described in Fourcroy et al with some modifications.Initially, extraction was carried out devoid of adding internal standards (IS) to recognize relevant compounds, such as these growing (or appearing) with Fe deficiency.This extract was also employed to check for the presence on the compounds utilised as IS and also other endogenous isobaric compounds that could coelute with them, given that in each circumstances there is going to be analytical interferences in the quantification method.The extraction was then carried out adding the following 3 IS compounds artemicapin C (Figure D), a methylenedioxycoumarin, for quantification from the coumarins scopoletin, fraxetin, isofraxidin and fraxinol; esculin (Figure A), the glucoside type of the coumarin esculetin, for quantification of coumarin glycosides; as well as the lignan matairesinol (Figure D), for quantification of coumarinolignans.Frozen roots (ca.mg) were ground in liquid PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21543800 N applying a Retsch M ball mill (Restch, D seldorf, Germany) for min and then phenolic compounds were extracted with ml of LCMS grade methanol, either alone or supplemented with of a IS solution (.artemicapin C, esculin and .matairesinol) by homogenization in the similar mill for min.The supernatant was recovered by centrifugation (g at C and min), and stored at C.The pellet was resuspended in ml of methanol, homogenized once again for min plus the supernatant recovered.The two supernatant fractions had been pooled, vacuum dried within a SpeedVac (SPDV, ThermoSavant, Thermo Fisher Scientific, Waltham, Massachusetts, MA, USA) and dissolved with of a resolution containing methanol and .formic acid.ExtractsFrontiers in Plant Science www.frontiersin.orgNovember Volume ArticleSisTerraza et al.Coumarins in FeDeficient Arabidopsis Plantswere filtered through polyvinylidene fluoride (PVDF) .ultrafreeMC centrifugal filter devices (Millipore) and stored at C until analysis.Phenolic compounds inside the nutrient solutions ( ml of resolution utilised for the growth of plants) have been retained in a SepPack C cartridge (Waters), eluted from the cartridge with ml of LCMS grade methanol, plus the eluates stored at C.Samples were thawed along with a aliquot was dried below vacuum (SpeedVac) alone or supplemented with of a IS option ( artemicapin C and matairesinol).Dried samples were dissolved in methanol and .formic acid to a final volume of , then analyzed by HPLCMS.No determinations may be made in nutrient options of Fesufficient plants because of the presence of Fe(III)EDDHA, that causes the overloading of C materials.Extraction of Cleomiscosins from Cleome viscosa SeedsCleomiscosins have been extracted from Cleome viscosa seeds (B T.
