SUPPORTING INFORMATION
Figure S1. Effect of salt stress on plant morphology
Figure S2. Effect of salt stress on photosynthetic performance,
chlorophyll fluorescence, chlorophyll content and ion contents of (A)
Responses of carbon exchange rate (CER) (µmol m-2s-1), (B) changes in maximal photochemical efficiency
of PSII (FV/Fm), (C) total chlorophyll content, (D) relative water
content in the leaves and roots of control and salt-treated plants (RWC)
(%), (E) Cl- ion content in the leaves and roots of
control and salt-treated plants, (F) Ca2+ ion content
in the leaves and roots of control and salt-treated plants, (G)
Na+ in the leaves and roots of control and
salt-treated plants, (H) and (I) variations in the ratios of
Na+/Cl- and
Na+/Ca2+ ratios in the leaves and
roots of control and salt-treated plants. Error bars represent the mean
± SD (n=6). Two-way ANOVA test was performed to measure P-values ns (not
significant) * (P<0.05), ** (P<0.01) and * * *
(p<0.001) respectively.
Figure S3. Illumination of Na+ ion
fluorescence in leaves of control and salt-treated P. pinnata .
Confocal images of leaves were stained with CoroNa-Green AM (green
color) and propidium iodide (red color). (A, B and C) cross sections of
leaves of control plants, (D, E and F), cross sections of leaves of 300
mM NaCl 1, 4 and 8DAS, (G, H and I) cross sections of leaves 500 mM NaCl
1, 4 and 8DAS respectively. Red arrows were drawn to measure
Na+ fluorescence intensity. Quantification of
Na+ fluorescence intensity more than 12 images were
pooled from five biological replicates in control and salt-treated
plants. Error bar represents the mean ± SD (n=12). Two-way ANOVA test
was performed to measure P-values ns (not significant) *
(P<0.05), ** (P<0.01) and * * * (p<0.001)
respectively.
Figure S4. Illumination of Na+ ion
fluorescence in roots of control and salt-treated P. pinnata .
Confocal images of roots were stained with CoroNa-Green AM (green color)
and propidium iodide (red color). (A, B and C) cross sections of roots
of control plants, (D, E and F), cross sections of roots of 300 mM NaCl
1, 4 and 8DAS, (G, H and I) cross sections of roots 500 mM NaCl 1, 4 and
8DAS, (J, K and L) magnified images of roots of control plants, (M, N
and O) magnified images of roots of 300 mM NaCl 1, 4 and 8DAS, (P, Q and
R) magnified images of roots 500 mM NaCl 1, 4 and 8DAS respectively.
White arrows were drawn to measure Na+ fluorescence
intensity. Quantification of Na+ fluorescence
intensity more than 12 images were pooled from five biological
replicates in control and salt-treated plants. Error bar represents the
mean ± SD (n=12). Two-way ANOVA test was performed to measure P-values
ns (not significant) * (P<0.05), ** (P<0.01) and * *
* (p<0.001) respectively.
Figure S5. Heat map of metabolite changes in leaves of P.
pinnata under salt stress. Log2 ratios of the relative
fold change values (1 to 12) of each metabolite is represented in the
form of a single horizontal row portioned with six columns of two salt
treatments (300 and 500 mM NaCl) and three different time points (1, 4
and 8DAS) respectively. Each metabolite value is an average of six
biological replicates represented with different color codes red or blue
according to the scale bar. The color scale indicates degree of
correlation.
Figure S6. Heat map of metabolite changes in roots of P.
pinnata under salt stress. Log2 ratios of the relative
fold change values (1 to 12) of each metabolite is represented in the
form of a single horizontal row portioned with six columns of two salt
treatments (300 and 500 mM NaCl) and three different time points (1, 4
and 8DAS) respectively. Each metabolite value is an average of six
biological replicates represented with different color codes red or blue
according to the scale bar. The color scale indicates degree of
correlation.
Figure S7. Representative figure of semi-quantitative PCR
analysis of transporter genes expression in leaves and roots of P.
pinnata under three different salinity stress conditions 0, 300 and 500
mM NaCl at 1, 4 and 8DAS. The expression of levels of each gene
normalized by using reference gene 18s rRNA.
Figure S8. The band intensities of transporter genes of
semi-quantitative PCR analysis (showed in Supplementary Figure
7) in leaves and roots of P. pinnata under three different
salinity stress conditions 0, 300 and 500 mM NaCl at 1, 4 and 8DAS
respectively.
Figure S9 . Relative mRNA expression levels of cell-wall enzymes
and antioxidant enzymes in leaves of P. pinnata under Salt Stress
Conditions. Log2 fold changes of CAT4, FeSOD, MnSOD,
Cu/ZnSOD1, Cu/ZnSOD1-like, POD, PAL1, PAL2, CAD2, and CAD6, in leaves
(A) and roots (B) of salt-treated plants of P. pinnata at 1, 4
and 8DAS respectively when compared to their corresponding controls.
Error bar represents the mean ± SD (n=6).
Figure S10. Representative figure of semi-quantitative PCR
analysis of cell-wall enzymes and antioxidant enzyme genes expression in
leaves and roots of P. pinnata under three different salinity
stress conditions 0, 300 and 500 mM NaCl at 1, 4 and 8DAS. The
expression of levels of each gene normalized by using reference gene 18s
rRNA.
Figure S11. The band intensities of CAD1 and CAD1-like genes in
leaves and roots of P. pinnata under three different salinity
stress conditions 0, 300 and 500 mM NaCl at 1, 4 and 8DAS respectively.
Table S1. Relative concentration and fold changes of major
metabolites in leaves of P. pinnata under three different
salinity stress conditions 0 (Control), 300 and 500 mM NaCl at 1, 4 and
8DAS. Six biological replicates were used to measure the relative fold
changes of each metabolite and two-way ANOVA test was performed to
measure P-values ns (not significant) * (P<0.05), **
(P<0.01) and * * * (p<0.001) respectively.
Table S2. Relative concentration and fold changes of major
metabolites in roots of P. pinnata under three different salinity
stress conditions 0 (Control), 300 and 500 mM NaCl at 1, 4 and 8DAS. Six
biological replicates were used to measure the relative fold changes of
each metabolite and two-way ANOVA test was performed to measure P-values
ns (not significant) * (P<0.05), ** (P<0.01) and * *
* (p<0.001) respectively.
Table S3. List of all 71 metabolites of 300 mM NaCl treated
leaves of P. pinnata at 1, 4 and 8DAS and their abbreviations
showed in figure 3 A-F.
Table S4. List of all 71 metabolites of 500 mM NaCl treated
leaves of P. pinnata at 1, 4 and 8DAS and their abbreviations
showed in figure 3 G-L.
Table S5. List of all 71 metabolites of 300 mM NaCl treated
roots of P. pinnata at 1, 4 and 8DAS and their abbreviations
showed in figure 4 A-F.
Table S6. List of all 71 metabolites of 500 mM NaCl treated
roots of P. pinnata at 1, 4 and 8DAS and their abbreviations
showed in figure 4 G-L.
Table S7 . List of primers were used in semi-quantitative-PCR,
RT-PCR analysis to measure mRNA expression.