INTRODUCTION
The combination of global climate change and dwindling freshwater supplies has increased the need for salt and drought tolerant crops. Among woody nut perennial crops, pistachio, a dioecious tree in the family Anacardiaceae, exhibits relatively high drought and salinity tolerance (Walker et al., 1987; Ahmad and Prasad, 2012). Thus, pistachio has emerged as a nut crop of increasing commercial interest in the USA and around the world (Ferguson et al., 2002; Karimi et al., 2009; Ahmad and Prasad, 2012). In laboratory and field conditions, thePistacia vera scions, grown on several Pistacia spp.rootstocks, can tolerate sodium chloride (NaCl) concentrations of up to 150 mM (Walker et al., 1987; Ferguson et al., 2002). In contrast, citrus, grape, and avocado are all characterized as relatively salinity sensitive tree crops, and grape and avocado can only tolerate up to 50 mM and 15 mM NaCl respectively (Bernstein et al., 2004; Mohammadkhani et al., 2016; Ahmad and Anjum, 2020).
Salinity tolerance is a complex trait that involves the coordination of several interconnected mechanisms to minimize tissue damage upon salinity stress. These mechanisms include minimization of salt ion entry and loading into the xylem, maximizing intracellular compartmentalization in tissue, and ion retrieval before it reaches the leaves and shoot tips (Tester and Davenport, 2003; Gonzalez et al., 2012; Gupta and Huang, 2014; Chen et al., 2018; Yang and Guo, 2018; Munns et al., 2020). One well-studied mechanism is the regulation of ion homeostasis via the Salt Overly Sensitive (SOS) pathway at the plasma membrane. The SOS1 , an H+/Na+antiporter, regulates Na+ efflux from the cytosol to the surrounding extracellular medium, and has been associated with salinity tolerance phenotypes in Arabidopsis and halophytes, a group of plants known for their ability to survive in high salinity environments (Shi et al., 2002; Yang et al., 2009; Ji et al., 2013; van Zelm et al., 2020). The SOS pathway may also coordinate signaling with other salinity stress response pathways, such as the high affinity potassium channel (HKT ) located at the plasma membrane, to maintain ionic homeostasis by mediating potassium (K+) and Na+ flux (Yang and Guo, 2018). The regulation of these pathways, for example HKT1, is considered one of the salinity tolerance determinants in several diverse species including Arabidopsis, grape, and potato (Henderson et al., 2018; Yang and Guo, 2018; Zhang et al., 2019).
Plants can maximize intracellular compartmentalization of salt in tissues by sequestering excessive salt ions within vacuoles to reduce cytosolic toxicity (Zhang and Blumwald, 2001; Gonzalez et al., 2012; Gupta and Huang, 2014; Munns et al., 2016; Guo et al., 2020). This process is mediated by the H+ /Na+antiporters, encoded by the NHX1/2 genes (Zhang and Blumwald, 2001; Bassil et al., 2019). Additional layers of complexity include post-translational modification of antiporter proteins and retention of ions in the vacuole, both of which are associated with salinity tolerance (Tester and Davenport, 2003; Munns et al., 2016; Wu et al., 2019). There are limited studies in woody nut perennial species that investigate salinity stress responses at the cellular level with structural analysis and imaging methodologies. However, evidence is emerging in citrus for a role of vacuolar sodium sequestration in decreasing toxicity and minimizing sodium transported to leaves (Storey and Walker, 1998; Gonzalez et al., 2012).
Plants can also minimize salt entry via the regulation of ionic uptake through blockage of apoplastic transport. Specifically, apoplastic barriers can block bypass flow, which would otherwise allow Na+ to enter the shoot through the transpiration stream (Chen et al., 2018). The endodermis and exodermis cellular barriers are the innermost and outermost layers of the root cortex, respectively (Enstone et al., 2003; Barberon, 2017). Their function as apoplastic barriers has been attributed to two notable features of their cell walls: 1) the Casparian strip and 2) the suberin lamellae (Enstone et al., 2003; Doblas et al., 2017). The Casparian strip diffusion barrier is a deposition of lignin along the radial and transverse cell walls of the endodermis and exodermis. It forms a hydrophobic apoplastic barrier forces all apoplastic transport into the more tightly regulated symplastic system (Naseer et al., 2012; Lee et al., 2013). In contrast, suberin lamellae, composed mainly of long chain fatty acids, impregnate the entire exodermis/endodermis cell wall (radial, transverse, and tangential), forming a hydrophobic barrier that may play a role in further regulating ion and water uptake (Enstone et al., 2003; Serra et al., 2009).
Salinity influences both the timing and extent of suberization in apoplastic barriers, which in turn can affect the entrance of salt into vascular tissue (Enstone et al., 2003; Chen et al., 2011; Byrt et al., 2018; Wang et al., 2020). Apoplastic barriers can control Na+ uptake and transport to shoots, as demonstrated in rice, where blockage of apoplastic transport using silicate reduced sodium uptake (Yeo et al., 1999). Inhibition of suberin biosynthesis, via knockdown of CYP86A1/HORST, a cytochrome P450 dependent fatty acid ω-hydroxylase, leads to increased levels of Na+concentrations in Arabidopsis shoots and roots (Wang et al., 2020). To date, there have been very few investigations regarding the development of apoplastic barriers in woody fruit and nut crop species. In citrus, preliminary studies indicate that suberin deposition may increase in response to salt in higher order roots, and higher suberin deposition in exodermis may be associated with lower sodium uptake (Rewald et al., 2012; Ruiz et al., 2016). Olive shows increased suberization in response to drought stress, which reduced root hydraulics (Tataranni et al., 2015). In Pistacia species, the contribution of apoplastic barriers to salinity tolerance has hitherto not been examined.
Studies on the effects of salinity on pistachio rootstocks have proposed multiple mechanisms that can contribute to salinity tolerance, such as rootstock ion sequestration, increasing osmolyte concentration in shoot and root, and sodium interception by xylem tissue, (Picchioni et al., 1990; Ferguson et al., 2002; Akbari et al., 2018; Rahneshan et al., 2018; Godfrey et al., 2019; Jamshidi Goharrizi et al., 2020). Sodium exclusion in stem vascular tissue, in combination with sequestration in roots, was postulated to decrease sodium transport to the leaves (Godfrey et al., 2019). Thus, a combination of multiple mechanisms working in tandem is likely to account for pistachio salinity tolerance.
Among the few studies focusing on salt tolerance in the rootstocks of woody perennial nut species, none have focused on fine roots, where the bulk of water and ion uptake occur, or in the root tips, where ions such as potassium may accumulate (McCully, 1995; McCully, 1999; Ranathunge et al., 2011). We used fluorescence microscopy to investigate the localization of sodium and the differentiation of the root endodermis and exodermis across a developmental gradient in salt treated pistachio rootstocks. Our results indicate that a combination of sodium sequestration and apoplastic barrier differentiation contribute to salinity tolerance in pistachio rootstocks, and that these responses are coordinated across a root developmental gradient.