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.