Both vacuolar sequestration and suberin deposition are implicated in high salt tolerance
In the light of climate change based drought conditions which have led to increased soil salinity, pistachio provides an attractive model to dissect mechanisms of salinity tolerance and abiotic stress response (Ahmad and Prasad, 2012; Jazi et al., 2016; Bailey-Serres et al., 2019). While most of the studies so far have focused on the overall physiological plant responses (Picchioni et al., 1990; Ferguson et al., 2002; Karimi et al., 2009; Godfrey et al., 2019), there has been no analysis at the cellular and structural level in pistachio roots. In order to determine salinity tolerance mechanisms in pistachio, we analyzed Na+ sequestration and apoplastic barrier differentiation across a root developmental gradient using fluorescence microscopy. Our study demonstrated a significant increase of vacuolar Na+ sequestration and levels of suberin in the endo/exodermis in the more salinity tolerant UCB1 compared to P. integerrima, after salt treatment, suggesting that both mechanisms are utilized in tandem for salinity tolerance in pistachios.
Salinity tolerance is a complex trait that includes coordination of maximizing salt ion exclusion, regulation of salt ion transport, and ion sequestration at the cellular, tissue, and organ level (Tester and Davenport, 2003; Yang et al., 2009; Chen et al., 2011; Gupta and Huang, 2014; Chen et al., 2018). While salt ion extrusion at the plasma membrane is important in many species (Yang et al., 2009; Ji et al., 2013; Chen et al., 2018), it does not provide complete salinity resistance, as significant amounts of Na+ still enter the roots. Thus, additional mechanisms must be employed to limit salt transport to the salinity sensitive leaves. Vacuolar sequestration of Na+ is one such mechanism that reduces the amount of salt reaching the shoot, therefore reducing salt toxicity (Zhang and Blumwald, 2001; Gonzalez et al., 2012; Gupta and Huang, 2014; Guo et al., 2020). The dominant role of vacuolar Na+sequestration over Na+ exclusion in salinity tolerance was recently demonstrated in barley. Screening of 45 barley accessions for salinity tolerance mechanisms, using a fluorescence microscopy approach, demonstrated a positive correlation of vacuolar Na+ sequestration with the salinity tolerant varieties (Wu et al., 2019).
UCB1 plants showed a higher vacuolar Na+ sequestration capacity compared to P. integerrima (Fig 3). It is likely that UCB1 is more efficient at vacuolar sequestration due to the increased activity of salt ion antiporters such as the NHX transporter family (Gupta and Huang, 2014; Bassil et al., 2019; Guo et al., 2020). Overexpression of NHX1 increases salt tolerance in diverse species such as wheat, rice, tomato and mung bean (Zhang and Blumwald, 2001; Moghaieb et al., 2014; Kumar et al., 2017; Zeng et al., 2018) and likely contributes to vacuolar sequestration in pistachio. Retention of Na+ in the vacuole is also regulated by the control of Na+‐permeable vacuolar channels, which mediate the back‐leak of Na+ into the cytosol (Isayenkov et al., 2010; Munns et al., 2016; Munns et al., 2020). Thus, control of expression or activity of these channels can also contribute to the increased vacuolar Na+ in UCB1.
Another plausible scenario for lower P. integerrima vacuolar sequestration is that P. integerrima may be less efficient at minimizing salt ion entry, which leads to Na+ levels exceeding its tolerance threshold, causing cell damage and loss of vacuolar sequestration capacity. Analyses of expression and activity of salt ion antiporters such as NHX, AKT1, and SOS1, along with other Na+‐permeable channels and biotic stress response markers can help dissect the prominent pathway in UCB1 (Yang et al., 2009; Gupta and Huang, 2014; van Zelm et al., 2020).