CAX1a TILLING Mutations Modify the Hormonal Balance Controlling Growth and Ion Homeostasis in Brassica rapa Plants Subjected to Salinity

: Salinity is a serious issue for crops, as it causes remarkable yield losses. The accumulation of Na + a ﬀ ects plant physiology and produces nutrient imbalances. Plants trigger signaling cascades in response to stresses in which phytohormones and Ca 2 + are key components. Cation / H + exchangers (CAXs) transporters are involved in Ca 2 + ﬂuxes in cells. Thus, enhanced CAX activity could improve tolerance to salinity stress. Using the TILLING (targeting induced local lesions in genomes) technique, three Brassica rapa mutants were generated through a single amino acidic modiﬁcation in the CAX1a transporter. We hypothesized that BraA.cax1a mutations could modify the hormonal balance, leading to improved salinity tolerance. To test this hypothesis, the mutants and the parental line R-o-18 were grown under saline conditions (150 mM NaCl), and leaf and root biomass, ion concentrations, and phytohormone proﬁle were analyzed. Under saline conditions, BraA.cax1a-4 mutant plants increased growth compared to the parental line, which was associated with reduced Na + accumulation. Further, it increased K + concentration and changed the hormonal balance. Speciﬁcally, our results show that higher indole-3-acetic acid (IAA) and gibberellin (GA) concentrations in mutant plants could promote growth under saline conditions, while abscisic acid (ABA), ethylene, and jasmonic acid (JA) led to better signaling stress responses and water use e ﬃ ciency. Therefore, CAX1 mutations directly inﬂuence the hormonal balance of the plant controlling growth and ion homeostasis under salinity. Thus, Ca 2 + signaling manipulation can be used as a strategy to improve salinity tolerance in breeding programs. phytohormone B. rapa plants key CAX1


Introduction
Saline soils represent 3.1% of the total land area of the Earth. Thereby, salinity is a serious issue for crops because it causes remarkable yield losses [1]. This problem has become more important over the last years and it is expected to be even more important in the future because of climate change [2]. Most crop species are affected by salinity including species from the Brassicaceae family, such as cabbage, broccoli, and rapeseed [3]. The most common and soluble salt compound is NaCl. The high concentration of Na + and Cl − ions in saline soils cause osmotic potential imbalances hampering water study aims to test whether changes in Ca 2+ signaling through BraA.cax1a mutations could modify the hormonal balance of the plant leading to improved growth under salinity.

Experimental Design, Treatments, and Plant Sampling
Treatments were applied 30 days after sowing and were maintained for 21 days. Plants received two different treatments: control (without NaCl added to the nutrient solution) and salinity (150 mM NaCl supplemented to the nutrient solution). The factors considered in the experiment were the salinity (S) and the mutation (M). The experimental design comprised a randomized complete block with 8 treatments, 3 trays per treatment, and 8 plants per tray, thus a total of 24 plants were grown for each treatment. At the end of the experiment, plant leaves and roots were rinsed, dried, and weighed to obtain the fresh weight (FW). Then, leaves and roots were lyophilized to determine the dry weight (DW) and a part of the lyophilized leaf material was used to determine the phytohormone concentrations. Nine independent replicates from each treatment (n = 9) were finally used for the analytical determinations.
2.3. Analysis of Na + , Ca 2+ , and K + Concentrations Ca 2+ , Na + , and K + were determined subjecting the leaf samples to a mineralization process by wet digestion [25]. Next, 150 mg of dry leaves were milled and mineralized with a combination of nitric acid and hydrogen peroxide at 30%. Then, 20 mL of Milli-Q water were added and element concentrations were measured using ICP-MS (X-Series II; Thermo Fisher Scientific Inc., Waltham, MA, USA).

Statistical Analysis
The mean and standard error of each treatment was calculated from the 9 individual data of each parameter analyzed. To assess the differences between treatments we performed a one-way analysis of variance (ANOVA) with 95% confidence. A two-tailed ANOVA was used to determine whether the NaCl treatment (S), the BraA.cax1a mutations (M), or their interaction (S × M) significantly influenced the results. Means were compared using Fisher's least significant differences (LSD). The significance levels were stated as * p < 0.05, ** p < 0.01, *** p < 0.001, or NS (not significant). A principal components analysis (PCA) was performed to assess relationships between treatments and all parameters analyzed. All statistical analyses were carried out using the Statgraphics Centurion 16.1.03 software.

Plant Biomass and Cation Concentration
Plants grown under salinity conditions presented a remarkable decrease in leaf and root DW ( Figure 1). However, BraA.cax1a-4 plants grown under salinity showed significantly higher leaf biomass in comparison to the other mutants and the parent line. Indeed, this mutant presented 41% higher leaf DW than R-o-18 plants (Figure 1a). Nonetheless, the four lines analyzed did not show significant differences in root DW under saline conditions (Figure 1b). Regarding cation concentrations, salinity reduced leaf Ca 2+ concentration in comparison to control conditions in all lines, but no differences were observed between lines under salinity conditions. As expected, NaCl application strongly increased Na + concentration in leaves. However, this increment was lower in BraA.cax1a-4 plants, which showed the lowest Na + concentration. Additionally, NaCl application reduced K + concentration, although this reduction was lower in BraA.cax1a-4 plants, which presented the highest K + concentration in comparison to the other genotypes. Consequently, BraA.cax1a-7 plants presented the highest Na + /K + ratio, followed by BraA.cax1a-12, R-o-18, and BraA.cax1a-4 plants. Specifically, BraA.cax1a-4 mutant plants showed 42% lower Na + /K + ratio than R-o-18 plants (Table 1).

Statistical Analysis
The mean and standard error of each treatment was calculated from the 9 individual data of each parameter analyzed. To assess the differences between treatments we performed a one-way analysis of variance (ANOVA) with 95% confidence. A two-tailed ANOVA was used to determine whether the NaCl treatment (S), the BraA.cax1a mutations (M), or their interaction (S × M) significantly influenced the results. Means were compared using Fisher's least significant differences (LSD). The significance levels were stated as *p < 0.05, **p < 0.01, ***p < 0.001, or NS (not significant). A principal components analysis (PCA) was performed to assess relationships between treatments and all parameters analyzed. All statistical analyses were carried out using the Statgraphics Centurion 16.1.03 software.

Plant Biomass and Cation Concentration
Plants grown under salinity conditions presented a remarkable decrease in leaf and root DW ( Figure 1). However, BraA.cax1a-4 plants grown under salinity showed significantly higher leaf biomass in comparison to the other mutants and the parent line. Indeed, this mutant presented 41% higher leaf DW than R-o-18 plants (Figure 1a). Nonetheless, the four lines analyzed did not show significant differences in root DW under saline conditions (Figure 1b). Regarding cation concentrations, salinity reduced leaf Ca 2+ concentration in comparison to control conditions in all lines, but no differences were observed between lines under salinity conditions. As expected, NaCl application strongly increased Na + concentration in leaves. However, this increment was lower in BraA.cax1a-4 plants, which showed the lowest Na + concentration. Additionally, NaCl application reduced K + concentration, although this reduction was lower in BraA.cax1a-4 plants, which presented the highest K + concentration in comparison to the other genotypes. Consequently, BraA.cax1a-7 plants presented the highest Na + /K + ratio, followed by BraA.cax1a-12, R-o-18, and BraA.cax1a-4 plants. Specifically, BraA.cax1a-4 mutant plants showed 42% lower Na + /K + ratio than R-o-18 plants (Table 1). Values are expressed as means ± standard error (n = 9). Bars marked with different letters indicate significant differences among genotypes based on the LSD test (p < 0.05). Asterisk (*) indicates significant differences between control and 150 mM NaCl treatments. Values are expressed as means ± standard error (n = 9). Bars marked with different letters indicate significant differences among genotypes based on the LSD test (p < 0.05). Asterisk (*) indicates significant differences between control and 150 mM NaCl treatments. Values are expressed as mg g −1 DW and differences between means (n = 9) were compared by Fisher's least-significance test (LSD; p = 0.05). Values with different letters indicate significant differences among genotypes. The levels of significance were represented by p > 0.05: NS (not significant), p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Phytohormone Concentrations
Salinity increased total CKs, total GAs, and provoked differential changes in the concentrations of other hormones in R-o-18 and mutant plants ( Figure 2; Table 2). Under saline conditions, BraA.cax1a-4 was the only mutant that showed significantly higher IAA levels (55%) in comparison to R-o-18 plants ( Table 2). Regarding GAs under saline conditions, BraA.cax1a-4 plants presented the highest GA concentrations. The other two mutants presented similar values than R-o-18 plants (Figure 2a). Particularly, BraA.cax1a-4 showed significant increments in all GAs (4-fold higher than R-o-18), while in BraA.cax1a-7 only GA1 increased in comparison to the parent line (Table 2). Concerning CKs, iP decreased in the BraA.cax1a-4 mutant in comparison to R-o-18, whereas incremented in BraA.cax1a-7 plants. Importantly, tZ increased in both BraA.cax1a-4 and BraA.cax1a-7 mutants, and its absolute concentrations were much higher than those of iP, leading to increased total CK content ( Figure 2b). ABA concentration was significantly higher in BraA.cax1a-4 plants (57%) in comparison to R-o-18 plants. All mutants showed higher ACC levels in comparison to the parent line. SA increased in BraA.cax1a-4 and BraA.cax1a-7 mutants, whereas JA concentration increased in BraA.cax1a-4 and BraA.cax1a-12 in comparison to the parental R-o-18 (Table 2).

Principal Component Analysis
A principal component analysis (PCA) was performed to detect general trends in the data and to evaluate the relationships among parameters. The first principal component (PC1) of the score plot clearly separated BraA.cax1a-4 from the rest of the lines and accounted for 50.55% of the variance within the data. The second principal component (PC2) separated BraA.cax1a-7 from the other lines and accounted for 23.73% of the variance (Figure 3a). The PCA loading plot revealed three clusters (Figure 3b). The first cluster associated leaf DW with K + , GA3, GA4, ACC, GAs, and ABA levels. The second cluster related Na + , Na + /K + ratio, Ca 2+ , and iP levels. Finally, the third cluster, grouped tZ, total CK, GA1, and SA concentrations (Figure 3b). mutants, and its absolute concentrations were much higher than those of iP, leading to increased total CK content (Figure 2b). ABA concentration was significantly higher in BraA.cax1a-4 plants (57%) in comparison to R-o-18 plants. All mutants showed higher ACC levels in comparison to the parent line. SA increased in BraA.cax1a-4 and BraA.cax1a-7 mutants, whereas JA concentration increased in BraA.cax1a-4 and BraA.cax1a-12 in comparison to the parental R-o-18 (Table 2).    Values are expressed as ng g −1 DW and differences between means (n = 9) were compared by Fisher's least-significance test (LSD; p = 0.05). Values with different letters indicate significant differences among genotypes. The levels of significance were represented by p > 0.05: NS (not significant), p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Discussion
Improved growth responses under salinity are associated with salinity tolerance. According to leaf DW results (Figure 1a), BraA.cax1a-4 presented higher performance under salinity in comparison to other lines. Several studies observed that a better Ca 2+ and K + nutrition and homeostasis provide salt tolerance due to Ca 2+ -associated signaling processes in response to stress and because it is a Na + antagonist [4,27]. In addition, Ca 2+ improves the accumulation of other nutrients and reduces Na + /K + ratio [11,13]. Although BraA.cax1a-4 plants did not present higher Ca 2+ concentration, they registered lower Na + and higher K + concentrations, leading to a better Na + /K + ratio (Table 1). A previous study showed that this fact could be due to BraA.cax1a-4 plants favors the transport of K + over Na + to the shoot from roots [28]. Hence, the modification of CAX1 activity could result in different ion accumulation, because of changes in Ca 2+ fluxes (but not absolute concentrations) in BraA.cax1a-4, improving Na + and K + homeostasis and thereby growth [29]. This is supported by the close relationship between leaf DW and K + concentration observed in loading plot analysis (Figure 3b). Alternatively, Ca 2+ is involved in modulating ROS levels that are generated by oxidative stress caused by salinity [5]. Thus, as observed in a previous study, the higher tolerance of BraA.cax1a-4 plants could also be related to this ROS modulation [28].
The possible alteration of Ca 2+ fluxes by BraA.cax1a mutations could affect the function of Ca 2+ sensors, such as calmodulins and protein kinases, that are crucial for hormone synthesis and signaling. Thus, the improved growth response under salinity of BraA.cax1a-4 plants could be also related to changes in the hormonal balance, as it has been proposed in the present study. Indeed, auxins, and particularly the active compound IAA, have crucial roles in stress signaling responses in B. rapa seedlings [3] and also participate in redox and antioxidative metabolism [9]. However, IAA concentration usually decreases and, thereby, senescence is promoted in plants grown under salt stress [30]. In the present study, we did not observe a significant reduction in IAA concentration. Thus, BraA.cax1a-4 was the line with the highest IAA concentration, which could contribute to the higher growth observed in this mutant under salinity conditions (Table 2). In fact, the greater IAA levels might enhance ROS detoxification under saline stress which could increase the tolerance to NaCl [31], as previously demonstrated in BraA.cax1a-4 mutants [28].
CKs regulate several physiological processes, promote plant growth, and play important roles in salinity tolerance [32][33][34]. A decrease in CKs usually is an early response to salt stress [10], although the contrary was also reported by Ghanem et al. [35]. These authors observed that CK concentrations increased in plants grown under salt stress as a response to increased salinity tolerance in tomato. This study agrees with our findings as we observed an increment in the CK concentrations in the plants subjected to salinity. Total CK concentrations significantly increased in the mutant, which presented the highest biomass under salinity, BraA.cax1a-4, compared to the parent line ( Figure 2b). However, their role in the control of growth of BraA.cax1a mutants seems to be limited since the PCA analysis revealed that CKs do not associate with any of the growth-related parameters recorded in this assay (Figure 3b).
Some studies have stated that GA accumulation in plants grown under abiotic stress provide salinity tolerance [36], whereas other studies have shown GA reduction because of repressor protein accumulation, leading to plant growth reduction [4]. Our results show that total GAs-especially GA3-markedly increased in BraA.cax1a-4 plants under salinity (Table 2, Figure 2a). GA3 is the main GA that regulates important steps in plant growth and development and alleviates salt stress [10]. Furthermore, Khan et al. [11] proved that the exogenous application of GA3 to linseed, alone or in combination with Ca 2+ , reduced the damage to membranes and improved water status. Therefore, this particular increment in GA3 and GA4 could also explain the higher biomass of the BraA.cax1a-4 mutants under salinity stress, as suggested by the close association between GAs and leaf DW in the PCA (Figure 3b).
Despite auxins, CKs, and GAs play a role in the response of the plants to salinity and other abiotic stresses, the phytohormone more broadly studied in relation to water and salinity stress is ABA, since this hormone is over-produced as a consequence of both stresses. Thus, genes related to ABA synthesis and accumulation are up-regulated by NaCl application, and in turn, ABA is the main hormone that activates salt responsive genes [37]. Our data reflects an increase in ABA concentration under salinity, which was especially strong in the BraA.cax1a-4 mutant ( Table 2). ABA triggers stress responses, such as water balance and osmotic stress regulation, and leads to stomatal closure to avoid excessive water loss [38]. This fact could be important in BraA.cax1a-4 plants where the high ABA levels provoke the closure of the stomata, thus enhancing water use efficiency (WUE) (data not shown). This is in agreement with previous studies in maize and tomato in which salinity-tolerant genotypes presented greater WUE and stomatal regulation, and lower Na + accumulation than the sensitive ones, associated with higher ABA concentrations [39,40]. Furthermore, Iqbal et al. [10] proved that Ca 2+ induces ABA accumulation, thus reducing Na + and Cl − content and increasing K + content. Therefore, ABA could contribute to better ion homeostasis via Na + /K + reduction, as observed in BraA.cax1a-4 plants (Table 1), maintain growth in plants grown under salinity conditions. This is further supported by the linkage among ABA, K + , and leaf FW shown in the PCA plot (Figure 3b).
Another hormone traditionally associated with stress responses is ethylene, especially in relation to leaf senescence and abscission. Salinity stress triggers the accumulation of the ethylene precursor ACC, leading to de-novo synthesis of ethylene, which induces cell death and leaf senescence, and reduces plant growth [9]. In our study, ACC concentration was higher in plants subjected to salinity which could contribute to biomass reduction (Table 2, Figure 1). Likewise, de la Torre-González et al. [31] observed that a salinity-tolerant tomato genotype presented much lower ACC concentration than the sensitive one under saline conditions. However, some studies proved that plants supplied with ACC or that overexpress ethylene-responsive factors presented tolerance to salinity stress [36,41]. Thus, all mutants evaluated in the present study showed a significant increase in ACC concentrations, and importantly, the BraA.cax1a-4 mutant presented the highest concentrations (Table 2) which may be associated with its improved growth-response due to better regulation of Ca 2+ homeostasis under salinity. This conclusion is additionally endorsed by the link of ACC with leaf FW and K + (Figure 3b). Indeed, the increased growth of BraA.cax1a-4 mutant under salinity stress could be partially associated with improved ethylene regulation of the high-affinity K + transporters, which increase K + tissue accumulation [41], as observed in this mutant (Table 1).
SA and JA regulate biotic stress responses but also are involved in regulating abiotic stresses, such as salinity, in cooperation with other hormones [12,42]. JA appears to provide tolerance to salt stress via plant signal transduction [7], whereas SA induces antioxidant defenses, protects photosynthesis, and reduces membrane damage [10,43]. Iqbal et al. [10] observed that JA reduces membrane depolarization and, thereby, K + loss. Hence, the highest JA concentrations of BraA.cax1a-4 and BraA.cax1a-12 (Table 2) could prevent K + leakage and contribute to the higher K + concentrations detected in these two mutants (Table 1). Additionally, this also may be explained by the lower lipid peroxidation levels previously observed in BraA.cax1a-4 and BraA.cax1a-12 plants [28]. Regarding SA, we observed a significant increment in SA concentration in all mutant plants grown under salinity in comparison to the parental R-o-18 plants (Table 2). Ku et al. [7] proved that increments in cytosolic Ca 2+ are a necessary step in SA accumulation in response to salinity. Thus, BraA.cax1a mutations could induce a higher SA accumulation through a greater cytosolic Ca 2+ response.

Conclusions
The present study demonstrates that the control of Ca 2+ homeostasis through BraA.cax1a mutants modifies the phytohormone balance of B. rapa plants controlling growth responses under salinity stress conditions. Specifically, the growth improvement of BraA.cax1a-4 mutant plants under salinity can be primarily associated with higher GA, IAA, and CK concentrations. Besides, ABA accumulation activates signaling stress responses under saline conditions, leading to better control of Na + /K + homeostasis. This study confirms the key role of the CAX1 transporter in phytohormone regulation and as a potential target for improving growth under salinity stress. However, further research is still necessary to mechanistically demonstrate the relationship between Ca 2+ fluxes and phytohormone accumulation in the control of the growth and productivity under salinity conditions.