Quantitative Metabolite Profiling in Maize

Maize or Arabidopsis material was ground to a fine powder by hand in a mortar pre-cooled with liquid N₂ or in a cryo-robot (Stitt et al., 2007 ), and stored at –80 °C. Samples were analyzed by LC-MS/MS and GC-MS, with authentic standards for accurate metabolite quantification, as in Heise et al. (2014) (link). We additionally analyzed aspartate, PEP, 2-phosphoglycolate (2PG), ribose-5-phosphate (R5P), and ribulose-5-phosphate+xylulose-5-phosphate (Ru5P+Xu5P) [see Supplementary Tables S1, S2 for the isotopomer-dependent MS parameters used for selected reaction monitoring (SRM) and the corconfig.cfg file used to correct for natural abundance; Heise et al., 2014 (link); Huege et al., 2014 ]. Amounts of the unlabeled form and each ¹³C isotopomers in maize samples are provided in Supplementary Table S3 and total contents in Supplementary Table S4. Total amounts of 3PGA and PEP were determined enzymatically using a Sigma-22 dual-wavelength photometer (Merlo et al., 1993 ) using freshly prepared extracts for PEP. ¹³C Enrichment and isotopomer distribution were calculated as in Szecowka et al. (2013) (link) (Supplementary Tables S5, S6). Active and inactive pools were calculated as in Supplementary Table S3, positional ¹³C enrichment (C4 and C1–C3 positions) of aspartate and malate as in Supplementary Tables S7, S8, and ¹³C amounts in metabolites as in Supplementary Tables S7, S8.