UCB1 shows vacuolar Na+ sequestration
In order to investigate the mechanism of salt tolerance, we visualized the cellular localization of sodium ions in live root sections with CoroNa Green, an indicator that exhibits an increase in green fluorescence emission intensity upon Na+ binding (Park et al., 2009; Gonzalez et al., 2012). Root developmental signals have several control points, and biotic stress has been shown to induce root differentiation closer to the apical meristem (Rost, 2011; Cajero-Sanchez et al., 2019). To assess the relationship between root maturity and salinity tolerance responses, Na+ localization was analyzed across a root developmental gradient, staged by type and extent of xylem formation (Fig 3a, b, Supp. Fig. 2). The characterization of the stages was established as follows: zone 0 represented the youngest (least mature) differentiated root region in which only protoxylem is present, zone 1 represented the region of intermediate development in which both protoxylem and metaxylem are present, zone 2 represented the most mature region, in which development of secondary xylem has been initiated (Fig. 3b, Supp. Fig. 2).
We observed sodium localization in the cortical parenchyma cell vacuoles of salt treated UCB1 seedlings (Fig. 3a). We confirmed the viability and physiological activeness of the cells in the root cross sections with fluorescein acetate staining, which measures both enzymatic activity (required to activate its fluorescence) and cell-membrane integrity (required for intracellular retention of their fluorescent product) (Supp. Fig. 3). Comparison of vacuolar staining across all developmental zones showed a consistent increase in Na+ positive vacuoles in UCB1 compared to P. integerrima (Fig. 3c, P<0.01 between genotype, P<0.05, between treatment, two-way ANOVA). A post-hoc Tukey’s HSD analysis verified the significant difference between control and treated UCB1 plants (Fig. 3c, P<0.05), and showed no difference in P. integerrimaseedlings (Fig. 3c, NS, two-way ANOVA). Notably, the highest number of Na+ stained vacuoles was observed in zone 1 of the UCB1 salt treated plants (Fig. 3d). This indicates an increase in Na+ sequestration under salt stress in the active growth zones of the root tips, before the onset of secondary growth. In contrast, P. integerrima showed minimal vacuolar localization of sodium with no significant increase after salinity treatment (Fig 3a, c, d). This suggests that root tip sequestration of Na+is likely a trait specific to UCB1. The majority of the cortical parenchyma cells of both untreated UCB1 and P. integerrima plants did not stain positive for Na+ (Fig. 3a,c,d) demonstrating that Na+ vacuolar sequestration was enhanced in salinity stress.
Co-localization with the established vacuolar red fluorescent marker SNARF-1 (Rosquete et al., 2019) demonstrated that CoroNa Green indeed exhibited vacuolar localization (Fig. 4a-c). Negative controls did not show cross talk between the acquisition spectra of the dyes (Fig. 4d-i), confirming signal specificity and true vacuolar Na+localization. Furthermore, CoroNa-Green and SNARF-1 signal were not affected by plant tissue autofluorescence, confirming the validity of our imaging methodology (Supp. Fig. 4).
Since the vacuolar localization of Na+ suggests sequestration in root tissue, we measured the Na+concentration in the roots of P. integerrima and UCB1 after one week of salt treatment. Although both UCB1 and P. integerrimaroots showed a significant Na+ increase after NaCl treatment (Supp. Fig. 5, two-way ANOVA, P <0.01 between treatment), the increase in UCB1 was significantly lower than that inP. integerrima (Supp. Fig. 5, two-way ANOVA, P=0.01). This corroborates earlier studies that UCB1 has lower Na+concentration in aerial tissues compared to P. integerrima(Ferguson et al., 2002). We also measured the K+ concentration, since it is an ion that is known to accumulate during salt stress as a result of the plant’s need to maintain ionic homeostasis (Chen et al., 2018; Ahmad and Anjum, 2020). We did not observe significant changes in the K+concentration in roots of either UCB1 or P. integerrima after treatment.
Cumulatively, our data demonstrated increased sodium sequestration in the vacuoles of UCB1 roots compared to P integerrima in response to salt treatment. However, the root Na+ concentration was higher in P. integerrima, suggesting higher uptake and accumulation of non-vacuolar Na+ in this salt sensitive genotype.