Fig. 5

Overview about the extraction and purification protocol. Samples are extracted with acidified MeOH (containing isotope labeled phytohormone standards and 4-methylumbelliferone). An aliquot is used as Fraction 1 for the analysis of amino acids, various carboxylic acids, high abundance 2nd metabolites (e.g., caffeoylputrescine, chlorogenic acid, nicotine and rutin) and sugars. The samples were diluted with aqueous solutions containing either ¹³C, ¹⁵N-labeled amino acids or sorbitol, as internal standards, before the analysis. The remaining extract was combined with the re-extract of the pellet and purified on two solid-phase extraction (SPE) columns (HR-X and HR-XC). Analytes were retained on the second HR-XC column until sequential elution. Fraction 2 was used for the analysis of acidic phytohormones (ABA, SA, AXs and JAs), as well as for various compounds of the phenylpropanoid pathway. The low abundance compounds from Fraction 2 were measured after an additional concentration step. The Fraction 3 (CKs) was also concentrated before analysis. AAs amino acids, ABA abscisic acid, AXs auxins, CA chlorogenic acid, CKs cytokinins, CP caffeoylputrescine, FA formic acid, GAs gibberellins, HR-X and HR-XC solid-phase extraction columns, JAs jasmonates, Lab AA mix algae extract containing ¹³C, ¹⁵N-labeled amino acids, SA salicylic acid