UCB1 shows increased suberin deposition at the endodermis and
exodermis
In addition to Na+ vacuolar localization, we observed
a slight increase in Na+ signal in the cell walls of
the endodermis and the exodermis of the salt treated plants (Fig. 3a).
Therefore, we investigated further whether the development of suberin
lamellae in these two cell layers is regulated as part of the salinity
stress response. We used a well-established methodology for suberin
detection via staining with the lipophilic fluorochrome Fluorol Yellow
(Lux et al., 2005; Kreszies et al., 2019) to quantify suberin deposition
in the exodermis and endodermis.
Higher suberin deposition was observed throughout in the apoplastic
barriers of UCB1 compared to P. integerrima, a pattern that was
maintained or emphasized under salt treatment (Fig. 5a). Of the two
barriers, UCB1 endodermis suberization showed a greater increase in
response to salt treatment than P. integerrima , though the
increase is statistically significant for both genotypes (Fig. 5b
P<0.05 for P. integerrima, P<0.01 for UCB1,
two-tailed t-test). In contrast, the exodermis of P. integerrimashows a significant increase in suberization to salt treatment that was
not observed in UCB1 (Fig. 5b, P<0.05 for P.
integerrima, NS for UCB1, two-tailed t-test). When we examined these
changes by developmental zones, we observed that in P.
integerrima, the only statistically significant increase in
suberization occurred in the exodermis of zone 1 (Fig. 5c,
P<0.05, two-tailed t-test). Interestingly, the increase in
suberization observed in UCB1 endodermis was statistically significant
in both zones 0 and zone 1, with the greatest difference observed in
zone 0 (Fig. 5d, P<0.01 for zone 0, P<0.05 for zone
1, two-tailed t-test).
We then compared the zone-specific responses between genotypes, in salt
and control treatments. In control conditions, we found the greatest
difference in zone 0 (Fig. 5e, P<0.01), which is the youngest
region possessing high developmental plasticity. While there was no
significant difference between UCB1 and P. integerrimasuberization in zones 1 and zone 2 of control plants, under salinity
stress all developmental zones showed significantly higher suberin
deposition in UCB1 compared to P. integerrima (Fig. 5f,
P<0.01 for zone 0, <0.05 for zone 1 and 2,
two-tailed t-test). This suggests a greater salt response and higher
developmental plasticity of cellular barriers in UCB1, which would
enable better control of apoplastic transport in all developmental
zones.
In order to further investigate the distinction between suberin
deposition and endodermis differentiation in the two genotypes, we
quantified the percentage of suberized cells in the exodermis and the
endodermis. While UCB1 showed a significant increase in the number of
cells suberized after salt treatment in both endodermis and exodermis of
(P<0.05 for exodermis and endodermis, two-tailed t-test),P. integerrima only showed a significant increase in the
endodermis (P<0.05, two-tailed t-test). Further analysis
revealed that there is a significant difference between genotypes in the
percentage of cells suberized in the endodermis, but not the exodermis
(P<0.01 between genotypes, P<0.001 between
treatment, two-way ANOVA). UCB1 contained a reduced number of suberized
cells in the endodermis layer compared to P. integerrima . This
shows that the higher suberin fluorescence intensity observed in UCB1
(Fig. 5e, 5f) is due to an increase in the amount of suberin deposited
per cell, and not the number of suberized cells in the endodermis.