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. 2). 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 (Choi et al., 2014),
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 (Reiter et al., 2011) peak
scores of q<0.01, the peak quality threshold for inclusion in
MSstats quantitative DIA data analysis.
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). Extended exposure resulted in 501
significantly upregulated and 481 significantly downregulated out of
3015 proteins quantified at MP in 85g/kg, 486 significantly upregulated
and 473 significantly downregulated out of 3015 proteins quantified at
MP in 105g/kg, and 311 significantly upregulated and 149 significantly
downregulated out of 3011 proteins quantified after ten weeks at 75g/kg.
The ten 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 constant salinity treatments and the second most highly
upregulated in the 14-day 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
14-day acclimations and the second most highly upregulated protein in
the extended treatments, with an average 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 MP, 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 which were above the critical salinity threshold.