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.