A
0.25
0.20
DRM1
no treatment
40mM
100mM
1M
0.15
target/reference ratio
0.10
0.05
0.00
0.35
DRM2
0.30
0.25
0.20
0.15
0.10
0.05
0.00
t0h
t6h
t12d
timepoint
B
No treatment
40mM NaCl
100mM NaCl
1M NaCl
Online Resource 5 Salt treatment assay optimisation
WT seeds were sterilised and stratified for 2 days in darkness at 4ºC. Individual seeds were sown onto ½ MS plates and grown for
6 days before being transferred to ½ MS plates supplemented with either 0 mM NaCl, 40 mM NaCl, 100 mM NaCl or 1 M NaCl
(T0).
A) Graphs show the target/reference ratio from RT qPCR for AtDRM1 and AtDRM2 over a salt treatment optimisation series in
WT plants. SD of technical replicates are represented in the error bars. Samples were normalised to the reference gene (ACTIN2:
At3g18780). AtDRM1 and AtDRM2 were detected in all samples and remained constant at all three time-points assessed with 0
mM salt treatment (control). AtDRM1 and AtDRM2 showed an increase in transcript expression levels with increasing
concentrations of salt, with the exception of AtDRM2 with 1 M NaCl treatment which showed no induction in the presence of salt
and remained at pre-treatment levels over the time-points assayed. Between 6h and 12 days treatment there was no difference in
expression levels for either gene. Seedlings were not sampled after 12 days with 1 M NaCl as this concentration was lethal.
B) WT plants after 12 days grown on varying concentrations of NaCl (0 mM NaCl, 40 mM NaCl, 100 mM NaCl, and 1 M NaCl)
across four different plates to reduce any inter-plate variation effects. WT survival was assessed 12 days after transfer to
treatment. WT plants showed a physical response to salt treatment, compared to no treatment, at all concentration of NaCl applied.
1 M NaCl was lethal for plants indicated by no additional growth after transfer and lack of green colour, whilst both 40 mM and
100 mM NaCl yielded viable plants indicated by growth and green colour. Based upon these observations 100 mM was selected
for subsequent salt treatment work.
DRM1 and DRM2 expression regulation: potential role of splice variants in response to stress and environmental factors in
Arabidopsis
Molecular Genetics and Genomics
Georgina M. Rae, Vladimir N. Uversky, Karine David, Marion Wood †
†
The New Zealand Institute for Plant & Food Research Limited, Mt Albert, Private Bag 92169, Auckland Mail Centre, Auckland
1142, New Zealand. [email protected]