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As knockout mutants of AtLeuC1 and AtIMD1, … As knockout mutants of AtLeuC1 and AtIMD1, two allelic lines of the respective genes [SALK_029510 (atleuc1-1) and SALK_065789 (atleuc1-2) for AtLeuC1, and SALK_063423 (atimd1-1) and SALK_ 069991 (atimd1-2) for AtIMD1] were used. In atleuc1-1, atimd1-1 and atimd1-2, expression of the corresponding gene was almost completely repressed. In atleuc1-2, transcript levels for AtLeuC1 were reduced but not completely repressed (Supplementary Fig. S1B). Knockout lines of AtBCAT4 (SALK_013627, bcat4-1) have already been reported (Schuster et al. 2006). As MAM1, MAM3 and AtLeuD1 knockout lines, SALK_012677 (mam1), SALK_004536 (mam3) and SALK_048320 (atleud1-1) were used. Homozygous lines of the T-DNA insertion mutants were selected by genomic PCR according to the T-DNA Express: Arabidopsis iSect Tool manual (http://signal.salk.edu/). Wild-type Arabidopsis thaliana (accession Colombia) and mutants (T-DNA insertion lines) were grown in a pre-fabricated room-type chamber at 22°C and a 16 h photoperiod in soil (PRO-MIX BX, Premier Horticulture, Rivière-du-Loup, QC, Canada) for seed collection, or on agar-solidified 1/2 Murashige–Skoog medium containing 1% sucrose for GSL analysis and widely targeted metabolomics. analysis and widely targeted metabolomics.
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