Analysis of gene expression
The cells were collected and shock-frozen in liquid nitrogen and ground with mortar and pestle (both heat-sterilied and then precooled) before extracting total RNA using Universal RNA Purification Kit (Roboklon, Germany). In case of leaf material, total RNA was isolated using the SpectrumTM Plant Total RNA Kit (Sigma, Deisenhofen, Germany) following the protocol of the producer. Gene expression was analysed at different time points after the incubation with 200 μM AlCl3. The extracted RNA was treated with a DNA-free DNase (Roboklon, Germany) to remove potential contamination of genomic DNA. The mRNA was transcribed into cDNA using the M-MuLV cDNA Synthesis Kit (New England BioLabs; Frankfurt am Main, Germany) according to the instructions of the manufacturer.
Steady-state transcript levels of the selected genes (PAL ,RS , STS , MYB14, PR1 and ICS ) were measured by quantitative real-time PCR (qRT-PCR) as described in Duan et al. (2015) using the oligonucleotide primers and PCR conditions given in Supplemental Table S1 . To compare the transcript levels between different samples, the Ct values from each sample were normalised to the value for the EF-1α internal standard obtained from the same sample. These normalised Ct values were averaged over each technical triplicate. The difference between the Ctvalues of the target gene X and those for the EF-1α reference R were calculated as follows: △Ct(X) =Ct(X)–Ct(R). The final result was expressed as 2–△△Ct(X). Each experiment was conducted in three biological replicates, i.e. independent experimental series.