Apoplastic barriers as a line of defense
UCB1 shows significantly higher levels of suberin than P. integerrima in both the endodermis and exodermis before salt treatment, and this difference was maintained after treatment (Fig. 5b-f). Furthermore, we detected a lower concentration of Na+in UCB1 roots compared to P. integerrima (Supp. Fig. 5), a pattern that was observed earlier in shoot tissue (Ferguson et al., 2002; Godfrey et al., 2019). One can conclude that UCB1 is more efficient at minimizing salt ion entry compared to P. integerrima, and that the high Na+ concentration inP. integerrima roots is a result of higher amounts of un-sequestered “free” Na+. Since the endodermis plays a role in the regulation of salt ion uptake by minimization of Na+ entry (Chen et al., 2018), and that salinity stress can induce suberin deposition (Enstone et al., 2003; Chen et al., 2011; Byrt et al., 2018; Chen et al., 2018), the superior salinity tolerance of UCB1 may be a result of the increased suberization at the apoplastic barriers. Higher baseline level of suberization can help reduce the exposure of root cells to ion toxicity at the initiation of salt treatment, increasing the overall health of the root cells during the critical early stages of salinity stress response. Such mechanisms have been proposed for rice and citrus (Ruiz et al., 2016; Vishal et al., 2019), and a similar mechanism may be taking place in UCB1.
The increase of suberin at the cellular barriers upon salinity stress was more pronounced in the endodermis and it was significantly higher in UCB1 across all developmental zones (Fig. 5b, e, f). Interestingly, the more sensitive genotype P. integerrima showed an increased number of suberized cells (Fig. 5g), but not increased suberin fluorescence intensity (across the entire exodermis/endodermis), suggesting that the initiation of the cellular barriers starts earlier in P. integerrima, but its rate of suberin biosynthesis and deposition is lower than UCB1. It is possible that some P. integerrima root cell types mature earlier compared to UCB1, and as a result, UCB1 maintains higher root plasticity in the same developmental zone. This is also a potential explanation for the increased number of lateral roots we observed in UCB1 compared to P. integerrima, since patterning of lateral root primordia is known to occur at the root tip and is highly responsive to abiotic stress (Koevoets et al., 2016; Julkowska et al., 2017; Dinneny, 2019).
Salinity tolerance and suberin deposition are energetically expensive for plants, and the plant must choose between the cost of cellular barrier enforcement and the cost of salinity stress (Tyerman et al., 2019). The increased basal level in suberin deposition at the endodermis in UCB1 suggests an early energy investment in enforcing salt exclusion from the vascular cylinder, and that this mechanism is more strongly utilized by UCB1 under salinity stress. Selection pressure for crop varieties with increased apoplastic barriers suberization has been shown for barley (Kreszies et al., 2020), suggesting that this energy investment is preferred in monocots. The popularity of UCB1 in pistachio breeding indicates a similar trend is taking place in woody perennial nut crops.