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.