Proteome regulation during salinity stress
Proteomic analysis was performed for seven treatments using the same DIA
assay library. The mass error threshold was <20 ppm for all
transitions in all samples and <10 ppm for the great majority,
and retention time reproducibility of the data was very high as well
(Supplementary fig. 3). A fold-change (FC) threshold of 2.0 was enforced
for all treatments in considering statistical significance based on the
coefficient of variation calculated with MSstats [33], providing at
least 0.8 statistical power for p-values <0.05. Furthermore,
the majority of transition peaks for all samples in this dataset had
mProphet [36] peak scores of q<0.01, the peak quality
threshold for inclusion in MSstats quantitative DIA data analysis.
Short-term acclimation to 85g/kg resulted in 234 significantly
upregulated and 171 significantly downregulated out of 2971 total
proteins quantified, while acclimation to 105g/kg yielded 348
significantly upregulated and 255 significantly downregulated out of
2972 total proteins quantified (Figure 3A, Supplementary table 1).
Exposure to LER resulted in 501 significantly upregulated and 481
significantly downregulated out of 3015 proteins quantified at 85g/kg,
and 486 significantly upregulated and 473 significantly downregulated
out of 3015 proteins quantified at 105g/kg. Long-term acclimation at
75g/kg resulted in 311 significantly upregulated and 149 significantly
downregulated out of 3011 proteins quantified.
The top five most highly upregulated and downregulated significant
proteins based on FC were determined for each treatment and compared
between treatments (Figure 3B). The most highly upregulated protein in
all extended treatments and the second most highly upregulated in the
short-term acclimations was inositol monophosphatase 1 isoform X1
(IMPase1-X1), which had a maximum upregulation in the extended 85g/kg
salinity treatment of 438 FC and an average FC increase of 225 across
all five treatments. Solute carrier family 12 member 2 isoform X1
(SLC12a2-X1) was the highest upregulated protein in both short-term
acclimations and the second most highly upregulated protein in the
extended treatments, with an average FC of 90 times greater across all
treatments. The most highly downregulated protein in four of the five
treatments was an uncharacterized protein, LOC100699110 isoform X1,
which was 1137 times lower in the extended 105g/kg exposure and 513
times lower on average across all treatments.
There was a large degree of overlap in significantly regulated proteins
in each treatment (Figure 3C). The greatest number of shared proteins
was between samples taken at LER, with 472 of the significantly
regulated proteins shared between the extended exposure at 85g/kg and at
105g/kg. The second largest group of overlapping proteins were those
which were significantly regulated in all treatments, including 161
proteins. The extended salinity treatments each had many proteins which
were only significantly regulated in one treatment, with 144 uniquely
significant proteins in the 85g/kg salinity, 110 in the 105g/kg
salinity, and 87 in the 75g/kg salinity. A total of 78 proteins were
significantly regulated in all four treatments above the critical
salinity threshold.