2.2. Plant Water Status and Physiological Monitoring of the Plants
Generally, during experiments, stem xylem water potential decreased with increasing water stress, especially in Carrizo and C35 citranges and
C. volkameriana (
Figure 2), with the latter exhibiting the lowest values. In detail, it was observed that Carrizo decreased the Ψ values from 199° DOYs until the end of the trial, whereas C35 significantly reduced the values with respect to the control only at the last detection (262° DOYs) at 66% and 50% Et
0 (59% and 64% less, respectively). During the trial Furr, citrumelo and
C. volkameriana showed a sensitive response to water stress, whilst Bitters, Carpenter and
C. macrophylla maintained similar values in all treatments (
Figure 2).
Overall, the net photosynthetic rate (A) monitored during the experiment showed a slight decrease in all rootstocks (
Figure 3). Carrizo citrange exhibited the highest A reduction with respect to the control at the end of the trial, precisely at 244° DOYs (−56.3% and −43.8%, respectively, for 66% and 50% Et
0) and at 262° DOYs (−59.3% and −66.6%, respectively, for 66% and 50% Et
0). The genotypes C35, Bitters, Carpenter, Furr and
C. macrophylla exhibited some statistical differences during the trial, even if no significant difference was observed at the end of the experiment in the two stressed treatments. Generally, transpiration rate (E) lowered during the experiment in all treatments (
Figure S1). Just at the end of the experiment, both treatments induced an increment of E in C35 and Citrumelo (
Figure 3), while Bitters and
C. volkameriana exhibited a reduction in E at the end of the trial with respect to the control in both stresses. Regarding the stomatal conductance (gs) a significant decrement of gs was observed in C35 citrange (−24.6% and −54.8%, respectively, for 66% and 50% Et
0) and in
C. volkameriana (−66.7% and −50.0%, respectively, for 66% and 50% Et0) in response to water stress (
Figure 3) at the end of the trial (
Figure S1). It was noted that Carpenter (−66.7), Furr (−60.0%) and Citrumelo (−33.3%) presented a significant reduction in the gs value at 50% Et
0, whereas
C. macrophylla showed an increase at 66% and 50% Et
0 (
Figure 3).
A significant reduction in the maximum potential quantum yield of PS2 (Fv/Fm) was recorded in C35 and Citrumelo at 66% Et
0 (
Figure S2), while a significant increase was noted in Bitters with respect to the control in both stresses (5.3% and 1.2%, respectively, for 66% and 50% Et
0).
2.3. Proline Accumulation, Malondialdehyde Determination and Antioxidant Enzyme Activities
At the end of the experiment, the accumulation of proline and malondialdehyde in the leaves and the antioxidant enzyme activities of the leaves were measured (
Figure 4).
A significant increase in proline, with respect to the control, was recorded in Furr and
C. volkameriana both at 66% and 50% Et
0 (
Figure 4). Carpenter showed a significant reduction only at 50% Et
0 with respect to the control (−34.0%), while Carrizo (−85.0%) and Citrumelo (−47.0%) showed a reduction at 66% Et
0. Finally, Carrizo, Citrumelo and C35 exhibited an increase with respect to the control only at 50% Et
0. No statistical differences among stressed and not-stressed plants were recorded in Bitters and
C. macrophylla.
As regards lipid peroxidation, Citrumelo and C. volkameriana showed no statistical differences at both stressed conditions compared to the control, whereas Carpenter and Furr reduced the MDA content at 66% Et
0 (−50.1 and −41.8%, respectively), but displayed similar values to the control at 50% Et
0. On the contrary, Carrizo citrange showed a significant increase in MDA values at both stresses (90.3 and 103.3%, for 66 and 50% Et
0), while C35 citrange exhibited a significant accumulation only at the maximum stress (102.1%) (
Figure 4). Finally, Bitters and
C. macrophylla showed an increased level of lipid peroxidation only at 66% Et
0 (130.8 and 94.6%, respectively).
As regards antioxidant enzymatic activities, different trends in each rootstock genotype were observed. All activities always showed similar values to those recorded in the control only in Furr, whereas a significant reduction was observed in Bitters (−68.6% for CAT, −36.7% for APX and 82.5% for SOD). In Citrumelo, all three enzymatic activities were higher, with respect to the control at both levels of stress, while in C. macrophylla, an increase was recorded only at higher levels of stress (93.7% for APX and 48.2% for SOD). In Carrizo, significant increases, with respect to the control, for CAT and SOD activities were recorded (305 and 60.6%, respectively), while a reduction was registered for APX values (−90.1 and −37.2% at 66 and 50% Et0). On the contrary, C35 showed an increase with respect to the control for CAT and APX. Carpenter reduced CAT activities but increased SOD at all levels of stress. Finally, C. volkameriana exhibited only an increase in SOD activities at both levels of stress.
2.5. Principal Component Analysis and Pearson’s Correlation Test
A principal component analysis (PCA) on the morphological, physiological, chemical and hormone parameters was performed to assess the differential response of several rootstocks under water stress (
Figure 6). The first two principal components accounted for 52.89% of the total variance (PC1 = 33.29%; PC2 = 19.6%). The main variables contributing to PC1 were A (0.310), followed by root length (0.333) and Ψ (0.273), while PC1 was negatively related with catalase activity (−0.343) and APX (−0.286). The PC2 was highly correlated with shoot length (0.360), IAA (0.358), ABA (0.330) and PA (0.323).
It was noticed that Furr (0.430), Bitters (0.360) and Carpenter (0.303) citrandarins were highly correlated with PC1, while Carrizo (−0.615) exhibited a negative relation with PC1. In fact, it was observed that citrandarins were greatly affected by physiological measurement, specifically by the net photosynthetic rate and xylem water potential (
Figure 2,
Figure 3 and
Figure S1). On the contrary, Carrizo showed a significant reduction in root length (
Table 1) and a great increase in catalase activity (
Figure 4). Carrizo citrange (0.495) showed a positive correlation with PC2, while
C. volkameriana (−0.485) and
C. macrophylla (−0.448) had a negative correlation with PC2. Carrizo exhibited a significant accumulation in ABA (
Figure 5), while
C. volkameriana exhibited a reduction in shoot length at 66% and 50% Et
0 (
Table 1).
Correlation coefficients of morphological, physiological, chemical and hormone parameters are shown in
Figure 7. In Carrizo citrange, the ABA value was positively correlated with PA (0.99), SA (0.87) and SAG (0.99), while it presented a negative correlation with IAA (−0.98) and Jalle (−0.92). PA exhibited a positive relationship with SA (0.83) while PA, JA, SA and SAG were negatively related with IAA (−0.99, −0.82, −0.74, −0.99, respectively) and with Jalle (−0.87, −0.99, −0.99 and −0.85). Water xylem potential was negatively related with JA (−0.50) and SA (−0.59). Net photosynthesis was positively correlated with all quantified hormones except Jalle (−0.81) and IAA (−0.99). The same trend was observed in the transpiration value, while gs exhibited a negative correlation with the IAA hormone (−0.91). It was observed that fluorescence had a high relationship only with hormones Jalle (0.95) and IAA (0.95). Catalase exhibited a high relationship with JA (0.79) and SA (0.85), while a strongly negative correlation was observed with Jalle, xylem water potential, MDA, shoot length and shoot/root ratio. SOD was negatively related with Jalle (−0.99), IAA (−0.98), Fv/Fm (−0.99), MDA (−0.86), shoot length (−0.94) and shoot/root ratio (−0.76). APX was mostly negatively related with gs, SPAD, leaf area and root length. Furthermore, MDA was positively related with Jalle, xylem water potential, Fv/Fm, shoot length and shoot/root ratio. Proline showed a strong negative correlation only with APX activity (−0.97). Furthermore, it was observed that shoot length and shoot/root ratio were negatively linked to all hormones except Jalle.
In C35 citrange, it was observed that abscisic acid was highly negatively correlated with PA (−0.99), CAT (−0.99), root length (−0.70), shoot length (−0.96) and shoot/root ratio (−0.97). PA was positively correlated with gs, CAT, shoot length and shoot/root ratio. In addition, JA was negatively associated with all enzyme activities, MDA, proline and leaf area. It was observed that Jalle, SA and SAG exhibited the same negative trend with gs, CAT, shoot length and shoot/root ratio. IAA was negatively correlated with SPAD (−0.97), enzyme activity, SOD (−0.97), APX (−0.97), MDA (−0.88), proline content (−0.97) and leaf area (−0.97). It was recorded that net photosynthesis, transpiration and fluorescence showed an inverse trend; the A value was highly correlated with SPAD, SOD, APX, MDA, proline and leaf area, while the E value and Fv/Fm were negatively related to these parameters. It was found that stomatal conductance was positively related to PA hormone (0.79), CAT (0.79) and shoot length (0.89). Proline content was negatively correlated with PA (−0.79), Ja (−0.80), IAA (−0.88), E (−0.96), Fv/Fm (−0.93), root length (−0.97), shoot length (−0.67) and shoot/root ratio (−0.95).
In the rootstock Bitters, it was observed that all hormones were correlated with each other except IAA which was negatively linked to ABA (−0.59), PA (−0.41), JA (−0.64) and SAG (−0.35). Furthermore, a very highly negative correlation was recorded between the hormones ABA, PA, JA, Jalle and SA and the values of SPAD and antioxidant activity, i.e., CAT and SOD. Xylem water potential and net photosynthesis exhibited the same negative trend with SPAD, CAT, SOD, MDA, leaf area and shoot/root ratio. The E value was negatively related only with IAA hormone (−0.90), leaf MDA content (−0.96), root length (−0.70) and shoot/root ratio (−0.79). Stomatal conductance and fluorescence showed the same negative trend with Jalle, IAA, APX and root length. SPAD values measured in leaves exhibited a positive correlation with enzyme activity (i.e., CAT and SOD), MDA and shoot/root ratio.
In Carpenter, it was recorded that ABA, JA, Jalle, SA, SAG and IAA were negatively correlated with physiological analyses, particularly with A, E and gs values. IAA was strongly negatively correlated with SOD (−0.92) and positively correlated with all other enzymatic measures. A negative correlation was recorded between xylem water potential and physiological measures, while Ψ was correlated with Fv/Fm.
Overall, it was observed in Furr that all hormones were positively correlated with each other. Water xylem potential was negatively correlated with ABA (−0.80), PA (−0.90), JA (−0.99), Jalle (−0.65), SA (−0.66), SAG (−0.26) and IAA (−0.80). All hormones were negatively correlated with SOD and APX. ABA, SA and IAA exhibited a large negative correlation with proline and leaf area. Transpiration was negatively correlated with water xylem potential (−1.00), fluorescence (−0.99), SOD (−0.99) and APX (−0.99). MDA had a positive relation with PA (0.89), Jalle (0.99), SAG (0.93) and SPAD (0.94).
Regarding Citrumelo, ABA showed a negative correlation with SA (−0.73). Abscisic acid was negatively correlated with all physiological activities, and it was highly negatively correlated with SOD (−0.90). PA, JA, Jalle and SAG were negatively correlated with MDA and Proline, while IAA showed a positive relationship with these measures. The A value was negatively related with MDA, Proline, leaf area, root and shoot length and shoot/root ratio. Furthermore, it was observed that fluorescence and SPAD had a negative correlation with A and E values.
Plants of C. macrophylla showed a positive correlation between hormones. ABA was negatively related to A (−0.83), E (−0.67), SPAD (−0.88), APX (−0.99), leaf area (−0.95), root length (−0.77) and shoot length (−0.55). PA, Jalle and Sa were negatively linked with E and gs values. Indeed, the xylem water potential was positively correlated with all parameters except A, E, gs and Fv/Fm.
In C. volkameriana was recorded that ABA exhibited a negative correlation with E, gs, SOD and MDA. Jasmonic acid was negatively related to SOD (−0.75), proline (−0.71), leaf area (−0.94) and shoot/root ratio (−0.74). Jalle was negatively related to catalase (−0.94) and ascorbate peroxidase (−0.99), while it was positively related to proline (0.92), leaf area (0.99) and shoot/root ratio (0.94). Photosynthesis was negatively linked to all hormones except JA and ABA. In addition, the A value was strongly negatively linked to SOD activity and leaf area.
