Figure legends
Figure 1. More UCB1 seedlings exhibited high tolerance phenotype compared to P. integerrima
(a) Representative high and low salinity tolerance phenotypes for the paternal genotype P. integerrima and hybrid line UCB1 after one week 100 mM NaCl treatment.
(b) Percentage of individuals in each genotype showing high, moderate, or low tolerance phenotypes. N=15 for P. integerrima , 22 for UCB1.
Figure 2. Short term salt treatment did not significantly reduce the growth of UCB1 or P. integerrima .
(a) Quantification of fresh weight after one week salt treatment showed that UCB1 seedlings were comparable in fresh weight to P. integerrima , and neither showed significant decreases with salt treatment in either total or tissue specific weight (NS, two-way ANOVA, N=7-9 plants per treatment per genotype).
(b) UCB1 exhibited a significantly higher number of lateral roots (LRs) per plant compared to P. integerrima (P<0.05 between genotypes, two-way ANOVA). This difference was increased after salt treatment (P<0.01 between treatment, two-way ANOVA, P=0.01, Tukey’s HSD, N=16-17 per treatment per genotype). Error bars = SEM.
Figure 3. UCB1 showed increased vacuolar sodium sequestration compared to P. integerrima .
(a) UCB1 and P. integerrima root tip section stained for Na+ showed increased number of vacuoles in UCB1 (indicated by arrowheads) after salt treatment and accumulation of Na+ at the exodermis and endodermis (3D construction was shown for zone 1 salt treated UCB1 to better illustrate Na+ vacuole localization).
(b) Diagram of root tip zones staged by xylem development. z0 = zone 0, z1 = zone 1, z2 = zone 2.
(c) Quantification of average number of vacuoles per cross section. (P<0.01 between genotype, P<0.05 between treatment, two-way ANOVA; * P=0.05 Tukey’s HSD.) (d) Quantification of average number of Na+ vacuoles staged by xylem development showed the highest vacuole count in zone 1 (NS, two-way ANOVA, N=15-51 sections/zone/genotype from 3-5 plants). Error bars = SEM.
Figure 4. Sodium staining in UCB1 showed co-localization with a vacuolar marker.
(a-c) Co-staining of Na+ with CoroNa green (a) and SNARF-1 for vacuole (b) showed co-localization in the vacuoles of salt-treated UCB1 root cross section (c, indicated by arrow). CoroNa Green staining alone (d-f), or SNARF-1 alone (g-i) do not show vacuolar co-localization.
Figure 5. UCB1 showed higher suberin deposition than P. integerrima in control and salt treated plants.
(a) Cross sections of root tips in both UCB1 and P. integerrimashowed increased suberization at exodermis (white arrow) and endodermis (red arrow) when stained with Fluorol Yellow 088. (b) Quantification of suberin fluorescence intensity (a. u.) across cross sections indicated increase in suberization in response to salt treatment in both exodermis and endodermis of P. integerrima and in endodermis of UCB1.
(c-d) Quantification of suberin fluorescence in zones staged by xylem development showed a peak in suberin increase after salt treatment in zone 1 of P. integerrima exodermis. In UCB1 both zone 0 and zone 1 showed an increase, with the greatest change occurring in endodermis of zone 0.
(e-f) UCB1 showed consistently higher suberization than P. integerrima in the exodermis of untreated plants for all zones, and higher suberization in zone 0 of endodermis. After salt treatment, UCB1 showed higher suberization than P. integerrima for all zones in both exodermis and endodermis.
(g) Salt treatment increased the percentage of suberized cells in the endodermis of P. integerrima and in both the exodermis and endodermis of UCB1 (* P<0.05, ** P<0.01, unpaired 2-tailed t-test) (N= 28-62 sections collected from 3-5 plants for B, G. N=3-28 sections per zone per genotype per treatment for C-F). Error bars = SEM.
Figure 6 . Model of UCB1 and P. integerrima root tip response to short-term salt treatment.
Under salinity stress, UCB1 plants were able to sequester excess Na+ in the vacuoles of cortical parenchyma of the root tips in contrast to P. integerrima. Under control conditions UCB1 deposited more suberin in cellular barriers. In addition, while both UCB1 and P. integerrima showed increased suberization of endodermis and exodermis in response to salt treatment, UCB1 showed higher suberization than P. integerrima.