Triazole

Caution! ⁹⁹Tc is a β-emitter (A = 635 Bq/μg [67 ], E_max = 290 keV); appropriate shielding and manipulation technics were employed during the synthesis and in all manipulations.
Pirimydine, 2,5-dimethilpyrazole, pyridazine, triazole were purchased from Sigma-Aldrich (Darmstadt, Germany). All reagents and solvents were qualified chemically pure and were not subjected to further purification.
The ammonium pertechnetate (ISOTOP RF) used in the work was recrystallized from bidistilled water (R ≥ 18 MΩ). The resulting white crystalline powder was especially pure and spectrally pure.

To analyze relative expression of genes involved in cutin and suberin biosynthesis, total RNA was isolated from the rosette leaves of 21-day-old soil-grown WT and ANAC046 transgenic plants using Triazole (Triagent, Sigma-Aldrich, St. Loius, MO, USA) method. For analysis in root tissues, WT and ANAC046 transgenic lines were grown hydroponically for 21 days and root tissues were harvested for RNA extraction. RNA samples were treated with RQ1 DNase (Promega, Madison, WI, USA) to digest any residual genomic DNA and were subsequently cleaned with the RNeasy Kit (Qiagen, Hilden, Germany). RNA samples were then subjected to cDNA synthesis using first-strand cDNA synthesis kit (Quantabio, Beverly, MA, USA). Reaction mixtures were prepared containing equal amount of cDNA (50–100 ng) of each sample and SYBR^® Green dye (Quanta) according to the manufacturer’s instructions and analysed using the Applied Biosystems 7500/7500 Fast Real-Time PCR System (Waltham, MA, USA). Actin 7 (ACT7) was used an internal control to normalize the expression values of target genes. Primers sequences for the analyzed genes are provided in supplementary Table S1.