sitl1 mutant enhances salinity stress insensitivity
sitl1 mutant showed significantly decreased root biomass and leaf
chlorophyll content when plants were grown in NS (Figure 4a-e). In
contrast, when plants were treated with NS containing 50 and 100 mM
NaCl, the sitl1 produced 1.22-fold, 1.19-fold, and 1.26-fold more
biomass in root, leaf sheath, and leaf blade than WT, respectively
(Figure 4b). Theses increased root and shoot biomass in the sitl1was also observed when plants were treated with DW containing 50 and 100
mM NaCl (Figure 4c). The sitl1 also had higher chlorophyll
content than the WT plant when plants were subjected to the treatment
with NS or DW containing 50 and 100 mM NaCl (Figure 4d,e). We further
confirmed that the sitl1 showed improved salinity insensitivity
when plants were irrigated with 50 mM NaCl solution under soil-grown
conditions but the sitl1 did not show drought stress
insensitivity (Figure S5). These results confirm that the sitl1have a higher ability to tolerate salinity stress but not drought
stress.
In order to visualize and quantitate the
H2O2 accumulations, we conducted
histochemical DAB staining and H2O2quantification analysis at 7 days after treatment with 0, 50, 100 mM
NaCl (Figure 4f-h). Under unstressed condition, no significant
differences were observed in the amounts of
H2O2 between the root tissues of thesitl1 and WT, whereas the sitl1 line showed a 1.47-fold
increase in H2O2 amount in leaf tissues
compared to the WT (Figure 4g-h). Under salinity stress condition, thesitl1 showed a 0.45- (50 mM NaCl) to 0.5-fold (100 mM NaCl)
decrease in root tissues, and 0.53- (50 mM NaCl) to 0.58-fold (100 mM
NaCl) decrease in leaf tissues, respectively, relative to the WT (Figure
4f-h).
Sodium ion concentration within the lateral roots was measured using the
cell-permeant CoroNa Green AM dye, which is a green-fluorescent
Na+-specific indicator that exhibits an increase in
fluorescence emission intensity upon binding Na+levels (Cho, Park, Kim, & Jang, 2010). Seven-day-old seedlings were
transferred to nutrient solution containing 50 mM NaCl for 3 h. The
salinity-treated WT plants showed strong fluorescence signals both in
the vacuoles and the cytoplasm of outer epidermis and inner cells,
whereas the sitl1 showed very little fluorescence signals, which
tightly sequestrated into the vacuoles in roots (Figure 4i).
In order to validate the ability of Na+ influx across
the plant plasma membranes, we used freshly isolated protoplasts from
leaf tissues because it was observed the difference in root development
of the sitl1 could alter the apoplastic permeability of
Na+ or the ability of root apopolastic barriers, which
block Na+ transport to shoot tissues in rice (Meier,
Kovalchuk, & Rose, 2006; Zhou et al., 2011). The salinity-treated WT
protoplasts showed obvious CoroNa Green AM staining, whereas the
protoplasts of the sitl1 showed very little staining (Figure 4j).
Quantitation of the CoroNa-Green AM intensity showed that the WT
displayed significantly higher fluorescence than the sitl1 in the
vacuole and the cytoplasm. Under unstressed conditions, there was no
significant differences in fluorescence intensity. However, under 50 mM
NaCl, the WT protoplast showed 6.95-fold and 4.15-fold higher
fluorescence intensity than the sitl1 , in the cytoplasm and in
the vacuoles, respectively (Figure 4k). These results demonstrate that
the improved salinity tolerance of the sitl1 caused major changes
such as reduced Na+ influx rate across the plasma
membrane in both root and leaf tissues.