Physiological mechanisms of ABA-induced salinity tolerance in leaves and roots of rice

Abscisic acid (ABA) plays a crucial role in response to abiotic stress as important small molecules in regulating metabolism. This study aimed to evaluate the ability of foliar spraying ABA to regulate growth quality at rice seedling stage under salt stress. Results demonstrated that salt stress strongly reduced all the growth parameters of two rice seedlings (‘Chaoyouqianhao’ and ‘Huanghuazhan’), caused prominent decrease in the levels of photosynthetic pigments (mainly in Huanghuazhan), photosynthesis and fluorescence parameters. Salinity treatment increased the concentration of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in roots, whereas significant decreased H2O2 was found in leaves of Huanghuazhan. Additionally, salinity triggered high Na+ content particularly in leaves and enhanced catalase (CAT) activities, ascorbate peroxidase (APX) and peroxidase (POD) activities of the two rice seedlings. Nevertheless, salinity-induced increased root ascorbic acid (AsA) and glutathione (GSH) levels while decreased in leaves, which depended on treatment time. Conversely, ABA application partially or completely mitigated salinity toxicity on the seedlings. ABA could reverse most of the changed physiological parameters triggered by salt stress. Specially, ABA treatment improved antioxidant enzyme levels and significantly reduced the Na+ content of two varieties as well as increased the K+, Mg2+ and Ca2+ content in leaves and roots. ABA treatment increased the hormone contents of 1-aminocclopropane carboxylic acid (ACC), trans-zeatin (TZ), N6-isopentyladenosine (IPA), Indole-3-acetic acid (IAA), and ABA in leaves of two rice varieties under salt stress. It is suggested that ABA was beneficial to protect membrane lipid peroxidation, the modulation of antioxidant defense systems and endogenous hormonal balance with imposition to salt stress.

Effects of exogenous ABA on the shoot morphological parameters under salt stress. Salinity treatment exhibited different degrees of reduction in all shoot morphological parameters during the treatment time course (Table 1). In 2-4 days, plant height shown a remarkable reduction in Q2 (on average by 8.69%) and Z2 (on average by 10.53%) when compared to Q0 and Z0, respectively; these trend became stronger in 6-10 days, which shown an average 16.26% decrease in Q2 and 11.44% decrease in Z2, respectively. When compared with salinity treatment alone (Q2 and Z2), ABA treatment restored the decreased plant height caused by salinity, which resulted in a slight increase in Q3 (on average by 1.69%) and Z3 (on average by 1.53%), respectively, from 6 to 10 days.
As shown in Table 1, salt stress prominently decreased the stem diameter in Q2 (on average by 12.20%) and Z2 (on average by 12.29%) compared to their respective control, in 6-10 days; however, these changed trend was reversed by application of ABA, which showed an average 6.35% increase in Q3 and 13.12% increase in Z3, respectively.
In comparison with Q0, salt stress treatment resulted in a slight reduction in leaf length and leaf area in Q2 during the treatment time course (Table 1). Notably, ABA + NaCl treatment (Q3) significantly increased the leaf area (on average by 34.58%) whereas a minor difference was found in leaf length during 2-10 days. Slight reduction was found in leaf length in Z2 when compared with Z0, from 2 to 6 days (Table 1), however, this trend www.nature.com/scientificreports/ became obvious from 8 to 10 days, which was decreased by 26.36% on average; notably, ABA + NaCl treatment (Z3) significantly increased the leaf length by 32.65% on average when compared to Z2 (8-10 days). Similarly, obvious reduction (on average by 36.04%) in leaf area was obtained in the presence of salinity (Z2) in 6-10 days, it was worth noting that ABA application strongly increased the leaf area by 112.62% on average in Z3 (6-10 days).

Effects of exogenous ABA on the root morphological parameters under salt stress.
In present study, we observed that salinity treatment (Q2 and Z2) exhibited an remarkable reduction in RL during treatment time course in comparison with Q0 and Z0, respectively; which decreased by 19.58% in Q2 and 24.84% on average in Z2, respectively (Table 2). In comparison with Q2, ABA restored the decreased RL caused by salinity in Q3 during 2-4 days (increased on average by 30.45%) and 8 days after salt stress (increased by 19.37%), respectively. Likewise, ABA treatment strongly increased RL in Z3 during 4-10 days (on average by 37.68%) when compared with Z2. As shown in Table 2, the RSA exhibited an obvious decline from 2 to 4 days (on average by 23.90%) whereas a slight decrease from 8 to 10 days (on average by 6.46%) in Q2 when relative to Q0, which were reversed by ABA from 2 to 4 days and 8 days after salt stress in Q3, elevated by 21.11% on average and 28.26%, respectively. However, salinity alone treatment significantly inhibited the RSA during the treatment time course (2-10 days) in Z2 when relative to Z0, which decreased by 25.20% on average. Notably, ABA + NaCl treatment (Z3) strongly elevated the RSL from 2 to 10 days (on average by 35.77%) when compared with Z2. Table 1. Effects of exogenous ABA on the shoot morphological parameters of five treatment time course in the presence of salinity. Q 0 treatment of water, Q 1 treatment with ABA, Q 2 treatment with NaCl, Q 3 treatment with ABA + NaCl in Chaoyouqianhao. Z 0 treatment of water, Z 1 treatment with ABA, Z 2 treatment with NaCl, Z 3 treatment with ABA + NaCl in Huanghuazhan. Datas are mean ± standard error of at least four replicates. Within each column, different letters indicate significant difference at the five percent significant level according to Duncan's multiple range tests. www.nature.com/scientificreports/ RV shown a high decrease from 2 to 4 days (on average by 14.51%) while a remarkable increase from 6 to 8 days (on average by 49.13%) in Q2 when relative to Q0; however, Q3 reversed the decreased RV caused by salinity from 2 to 8 days, though it was non-significant; notably, Q3 significantly increased RV at 10 days after salinity treatment (by 18.38%), when in comparison with Q2. In contrast to Chaoyouqianhao, RV exhibited a decrease trend during the treatment time course (on average by 26.06%) in Z2 when compared to Z0, whereas Z3 strongly enhanced RV from 8 to 10 days (on average by 81.29%).

Shoot morphological index Treatments
The results obtained from Q2 shown that salinity slightly enhanced the growth of RAD (on average by 7.08%) from 2 to 10 days while Q3 slightly increased it (on average by 2.71%) from 4 to 8 days. Additionally, Z2 exhibited an average 6.1% decrease in RAD when compared to Z0, while Z3 restored it (on average by 9.72%) from 8 to 10 days.
Effect of exogenous ABA on salt stress with respect to seedling biomass. Salinity treatment exhibited a different trend in shoot dry weight, root dry weight, total dry weight and root to shoot ratio between Chaoyouqianhao and Huanghuazhan ( Fig. 2a-h). When in comparison with the control, salinity treatment (Q2) decreased the shoot dry weight whereas Z2 showed a stronger decrease from 6 to 10 days, which were slightly reversed by Q3 and obviously reversed by Z3 (Fig. 2a, b), respectively. Q2 displayed a downward to an upward trend in root dry weight from 4 to 10 days when compared with Q0, whereas Q3 slightly increased root dry Table 2. Effects of exogenous ABA on the root morphological parameters of five treatment time course in the presence of salinity. RL indicates root length, RSA indicates root surface area, RV indicates root total volume, RAD indicated root average diameter. Q 0 treatment of water, Q 1 treatment with ABA, Q 2 treatment with NaCl, Q 3 treatment with ABA + NaCl in Chaoyouqianhao. Z 0 treatment of water, Z 1 treatment with ABA, Z 2 treatment with NaCl, Z 3 treatment with ABA + NaCl in Huanghuazhan. Values are mean ± standard error of four replicates (n = 4). Within each column, different letters indicate significant difference at the five percent significant level according to Duncan's multiple range tests. www.nature.com/scientificreports/ weight (on average by 8.77%) during the treatment time course (Fig. 2c). On the contrary, salinity treatment (Z2) strongly inhibited root dry weight (on average by 19.09%) during the treatment time course when compared to Z0, whereas Z3 prominently elevated it from 2 to 4 days (28.50%) and 10 days (65.12%) after salt stress, respectively (Fig. 2d). No significance was found in total dry weight among Q0, Q2 and Q3 (except at 8 days). Nevertheless, Z2 resulted in a significant decrease in total dry weight (on average by 18.86%) while Z3 strongly increased it (on average by 25.10%) in 2-10 days, in comparison with Z0 and Z2, respectively (Fig. 2f). As shown in Fig. 2g, when in comparison with Q0, Q2 exhibited a slight increment in root to shoot ratio (on average by 8.18%), which was increased by ABA + NaCl treatment (Q3) (on average by 13.95%) during 6-8 days. With regard to Huanghuazhan, Z2 significantly reduced root to shoot radio (13.44% on average) from 2 to 4 days Figure 2. Effect of ABA with leaf sprayed on shoot dry weight (a, b), root dry weight (c, d), total dry weight (e, f) and root to shoot ratio (g, h) of two rice cultivar seedlings under salt stress. Values are shown as the mean ± standard and derived from four replicates (n = 4). Bars indicate standard errors. Different letters above the bars indicate significant differences at the five percent significant level according to Duncan's multiple range tests.    (Fig. 4). However, ABA + NaCl treatment (Q3) showed a strong improvement in Pn (15.44%) in the presence of saline during 8 days of salt stress compared with Q2, while Z3 showed a stronger improvement (on average by 24.62%) in 2-8 days when compared with Z2. Similar to Pn, ABA showed an improvement in Gs with imposition of saline in comparison with Q2 at 2 days (46.48%) and 6 days (15.69%) of salinity treatment, respectively; while Z3 improved the Gs in 2-8 days (on average by 22.59%) when compared with Z2.
There were minor differences in Ci in the two varieties in NaCl + ABA treatments (Q3 or Z3) when compared with salinity treatments (Q2 or Z2) during treatment time course, except at 8 days of treatment in Q3 (an increase of 18.18%) and 6 days of treatment in Z3 (an increase of 14.20%) when compared with their respective control.
Moreover, ABA exhibited a different degree of enhancement in Tr in the presence of saline condition during 2-8 days, and the peak value of Tr was recorded at 6 days, which increased by 16.91% of control (Q2). Correspondingly, NaCl + ABA treatment (Z3) reversed the decreased Tr (on average by 18.05%) caused by saline during 2-8 days, when in comparison with saline alone treatment (Z2).

Effects of exogenous ABA on fluorescence parameters in rice seedlings under salt stress.
To assess the performance of PS II, chlorophyll fluorescence technique was used to determine some fluorescence parameters. Results showed that no significant difference between salinity treatment and the control group in Fv/Fm, Fv/Fo and Fm/Fo in both of two rice cultivars during the treatment time course (Table 3). Interestingly, Fv/Fm, Fv/Fo and Fm/Fo declined due to salinity treatment when compared with the control; conversely, NaCl + ABA treatment (Q3 or Z3) significantly improved Fv/Fm, Fv/Fo and Fm/Fo in 8 days after salinity.
As showed in Table 3, Q2 decreased the Qp when compared with Q0 during 2-4 days and 8 days of salinity treatment; however, Q3 showed a remarkable increase (41.98%) in 8 days. No significant difference was observed among all the treatments in Huanghuazhan during the treatmrent time course except 2 days after salt stress. Similarly, no significant difference was found among all the treatments in Chaoyouqianhao during 2-10 days, though Q2 strongly increased NPQ when compared with Q0 and it was reversed by Q3 when compared with Q2, in 6 days and 10 days after salt stress. Likewise, when in comparison with Z0, Z2 showed a significant increase (on average by 118.77%) in NPQ during 4-6 days. In contrast, Z3 restored the increased NPQ caused by NaCl (on average by 22.14%). In comparison with Q0, salinity treatment (Q2) resulted in a slight reduction in Qy, while Q3 improved Qy during the treatment time course except 6 days (Table 3). A decline in terms of Qy was observed in Z2 (on average by 6.57%) when compared with Z0 whereas Z3 improved it (on average by 7.67%) during 4-10 days.
Rfd is able to characterize the leaf potential photosynthesis quantum transformation efficiency. Results showed that salinity treatment (Q2) reduced Rfd when compared with Q0, whereas Q3 reversed the reduction caused by NaCl in 2-4 days and 8 days. Notably, salinity treatment (Z2) displayed an upward to a downward trend in Rfd from 2 to 8 days in comparison with their respective control; however, Z3 exhibited a minor difference in Rfd during 6-8 days.

Effects of exogenous ABA on ion homeostasis in rice seedlings under salt stress. Salt stress
significantly increased the contents of Na + in leaves and roots of two rice cultivars, which increased by 863.82 and 207.65%, respectively in Chaoyouqianhao and 1299.36 and 67.72%, respectively in Huanghuazhan (Fig. 5a, b). Compared with Q2, ABA + NaCl (Q3) significantly reduced the Na + content (by 8.31% in leaves and 23.54% in roots, respectively) and Z3 significantly reduced it by 39.85% in leaves, whereas minor difference was found in roots when compared with Z2.
The contents of K + in leaves and roots showed a significant decrease in salt-stressed rice seedlings when compared with non-stressed rice seedlings in terms of Chaoyouqianhao. However, Q3 significantly restored the decreased K + content caused by salt stress (Fig. 5c, d). Similarly, Z3 significantly increased it in leaves (by 18.03%) and roots (by 4.05%) of Huanghuazhan. The K + /Na + ratio in leaves and roots of the two rice cultivars exhibited a strongly decrease in presence of saline condition, due to the increase of Na + and the decrease of K + content, while ABA + NaCl treatment increased K + /Na + ratio in leaves and roots under saline condition (Fig. 5e, f).
To further clarify the regulation of ABA on mineral homeostasis under salt stress, the contents of two important ions Mg 2+ and Ca 2+ were measured ( Fig. 5g-j). Results showed that salinity treatment (Q2) significantly reduced Mg 2+ content in leaves (by 12.42%) and roots (by 19.48%) in comparison to their respective control (Q0). The decrease of Mg 2+ content was reversed by ABA + NaCl treatments (Q3), which increased by 25.43% in leaves and 13.82% in roots, respectively. Likewise, the decrease of Mg 2+ content was observed in leaves but not in roots of Huanghuazhan under salt stress; nonetheless, Z3 exhibited a significant increase in the two tissue. Interestingly, the content of Ca 2+ in roots significantly increased in Chaoyouqianhao under saline condition. Conversely, Z2 significantly reduced the Ca 2+ content in roots when compared with Z0. Nevertheless, Q3 or Z3 showed an enhancement in the content of Ca 2+ content, when compared with saline alone treatment (Q2 or Z2). in salinity treatment in both of two rice cultivars (Fig. 6a, b). Compared to Q0, MDA contents showed a significant increment in Q2 in roots (on average by 18.78%) from 2 to 4 days and in leaves (19.58%) at 8 days, respectively; however, no significance was found between Q3 and Q2 in MDA content of roots during the treatment time course while Q3 significantly reduced it in leaves compared with Q2 at 8 days (Fig. 6e). By comparison, Z2 strongly increased MDA level in roots (on average by 27.26%) during the treatment time course in comparison with Z0, which significantly decreased by Z3 (on average by 26.33%) from 4 to 8 days. Notably, Z2 showed a significant increase in MDA of leaves during 4-10 days except 8 days after salt stress, whereas Z3 reversed the increased MDA content (Fig. 6f).
The concentration of H 2 O 2 in roots in all treatments during 2-4 days were higher than that of 6-10 days in general (Fig. 6c, d). Salinity treatment (Q2) shown a prominent elevation in H 2 O 2 concentration in roots in the initial 2 days when relative to Q0, which was reversed by ABA in Q3; however, no significance was found among Q0, Q2 and Q3 from 6 to 10 days (Fig. 6c). The concentration of H 2 O 2 in leaves under salt stress significantly increased (on average by 26.63%) in Q2, which was reversed by NaCl + ABA (Q3) during 2-8 days (Fig. 6g). In comparison with Z0, H 2 O 2 in roots showed a strong increase in 2-10 days after salt stress; however, Z3 completely reversed (on average by 36.93%) the increased H 2 O 2 caused by salinity only from 4 to 10 days (Fig. 6d). It was of note that the concentration of H 2 O 2 in leaves significantly decreased in presence of salinity (Z2), whereas Z3 further decreased it, during 2-10 days except at 4 days.
Effects of exogenous ABA on the antioxidant enzyme activities in rice seedlings under salt stress. The CAT activities in roots showed increasing trends in the two rice cultivars from 2 to 6 days (Fig. 7a,   b). When in comparison with the control (Q0), Q2 caused a strongly increase in CAT (on average by 90.50%) whereas Q3 further increased it by 16.01% on average, from 4 to 8 days. Notably, salinity treatment (Z2) showed a 34.60% decrease in CAT activities compared with Z0, whereas Z3 strongly reversed (223.78%) the decreased CAT caused by salinity at 8 days. Similarly, CAT activities showed a strong increase in Q2 (on average by 14.85%) and Z2 (on average by 20.11%) from 6 to 10 days in leaves whereas no significance was found in ABA + NaCl treatment (Q3 and Z3) (Figs. 7k and l).
In comparison with Q0, salt stress treatment (Q2) resulted in a remarkable increase in APX activity in roots (on average by 41.21%) while Q3 weakly reversed it during 2-6 days of salt stress (Fig. 7c). Besides, the APX activity in roots shown no significance among Z0, Z2 and Z3 during the treatment time course except at 6 days (Fig. 7d). In comparison with the controls, APX activity in leaves displayed a stronger increment than that in roots in salinity treatment alone during the treatment time course (Fig. 7k, l). Notably, Q3 further enhanced APX activity (on average by 10.55%) in 4-6 days whereas Z3 further enhanced it (on average by 8.17%) in 2-4 days in comparison with their respective control.
The average activities of POD in roots remained relatively stable between the two rice cultivars (Fig. 7e, f). When relative to the controls (Q0 and Z0), both salinity (Q2 and Z2) and ABA + NaCl treatments (Q3 and Z3) significantly elevated POD activities from 4 to 10 days. POD activities in leaves showed a significant increase in Q2 when relative to Q0 from 2 to 6 days, and ABA treatment further increased it (9.60%) at 6 days (Fig. 7o). Similarly, Z2 significantly enhanced the POD activity in leaves during the treatment time course when compared to Z0, whereas Z3 further enhanced it from 2 to 4 days (Fig. 7p).
Under saline condition (Q2), SOD activity in roots exhibited a gradual increase trend (on average by 6.17%) during the treatment time course in comparison with Q0; however, no significance was found between Q3 and Q2 in general (Fig. 7g). Similarly, Z2 showed an average 8.10% increase in SOD activity in roots when compared with Z0 during 2-6 days, whereas a slight average 1.75% decrease in it from 8 to 10 days. However, no significance was found between Z3 and Z2 (Fig. 7h). In comparison with the control, salt stress significantly decreased the SOD activity in leaves at 4 days, which was restored by ABA in the ABA + NaCl treatment (Fig. 7q). The SOD activity exhibited a strong decrease when compared with the normal condition (Z0) at 8 days whereas Z3 showed a significant increment in it in comparison with Z2 (Fig. 7r).
In comparison with the controls (Q0 or Z0), salinity treatment showed a strong decrease in PAL activity in roots (on average by 34.11 % in Q2 and 27.95% in Z2, respectively) from 2 to 10 days (Figs. 7i and j). Obviously, Q3 gradually increased the PAL levels (on average by 45.51%) in 2 to 8 days relative to Q2 and the maximum range was observed at 8 days, whereas Z3 significantly enhanced the PAL activity (on average by 49.09%) in 4 to 10 days compared with Z2, and the maximum range was recorded at 6 days. As showed in Fig. 7s, Q2 decreased Table 3. Effects of exogenous ABA on fluorescence parameters of five treatment time course in the presence of salinity. Fv/Fm indicates maximal photochemical efficiency of PS II, Fv/Fo indicates potential activity of PS II, Fm/Fo indicates electron transfer case of PS II, NPQ indicates non-photochemical quenching coefficient, Qp indicates the coefficient of photochemical fluorescence quenching, Qy indicates effective photochemical efficiency of PS II, Rfd indicates fluorescence decrease ratio. Q 0 treatment of water, Q 1 treatment with ABA, Q 2 treatment with NaCl, Q 3 treatment with ABA + NaCl in Chaoyouqianhao. Z 0 treatment of water, Z 1 treatment with ABA, Z 2 treatment with NaCl, Z 3 treatment with ABA + NaCl in Huanghuazhan. Datas are mean ± standard error of at least four replicates. Within each column, different letters indicate significant difference at the five percent significant level according to Duncan's multiple range tests. www.nature.com/scientificreports/ the PAL activity in leaves in 4 to 6 days and 10 days when compared with Q0; notably, Q3 exhibited a significant increase in PAL from 4 to 10 days. No significance was found in PAL activity between Z0 and Z2, whereas Z3 prominently increased it at 2 days and 10 days, when compared with Z2 (Fig. 7t). www.nature.com/scientificreports/ Effects of exogenous ABA on the activities of AsA and GSH contents in rice seedlings under salt stress. The levels of AsA in roots displayed a significant increment in Q2 (by 85.89%) and Z2 (by 237.39%) compared with Q0 and Z0, respectively; which was further enhanced by ABA in Q3 (by 37.16%) and Z3 (by 14.30%), respectively in the initial 2 days of salt stress (Fig. 8a, b). Notably, the AsA contents shown increasing trends in Q2 from 6 to 10 days, which increased by an average 39.60% in comparison with Q0; moreover, ABA + NaCl treatment further increased AsA contents (on average by 24.12%) when compared with Q2 in Chaoyouqianhao. Conversely, salinity treatment (Q2 and Z2) decreased the AsA contents when compared with their respective controls, which decreased by 2.53% and 10.51%, respectively at 8 days (Figs. 8e and f); ABA + NaCl completely reversed the decreased AsA contents caused by NaCl (increased by 9.03% in Q3 and 17.45% in Z3). The GSH contents in roots showed an increasing trend during the treatment time course (Fig. 8c, d). Compared with Q0, Q2 displayed minor differences in GSH levels (on average by 5.25%), whereas Q3 significantly reduced the levels of GSH (on average by 44.03%) in 6-8 days, relative to Q2. Likewise, an average 14.13% increase in GSH levels was observed in Z2, which was reversed by ABA in Z3 (on average by 18.5%) in 6-8 days, reached to a significant level. Under salinity alone treatment, GSH contents in leaves displayed an opposite trend between the two rice cultivars (Figs. 8g and h). When compared with Q0, Q2 significantly enhanced GSH contents (on average by 19.44%) in 6-8 days, whereas the levels of GSH showed minor differences between Q3 and Q2. However, Z2 significantly decreased GSH contents (on average by 20.21%) in 6-8 days, whereas Z3 showed a strong increase in GSH contents (on average by 14.97%) in relative to Z2.

Scientific Reports
Effects of exogenous ABA on the levels of soluble protein in rice seedlings. The soluble protein contents in roots exhibited increasing trends from 4 to 8 days of the two rice cultivars (Fig. 9a, b). When compared with the controls (Q0 or Z0), salinity treatment exhibited an average 16.05% increase in Q2 and 12.16% in Z2, respectively, which were further enhanced by ABA in Q3 (on average by 8.41%) and Z3 (on average by 7.12%), respectively in 4-8 days.
Salinity alone treatment (Q2 or Z2) significantly increased the soluble protein contents in leaves in comparison with their controls, and Q3 and Z3 further enhanced the levels of soluble protein by 7.32% and 1.69%, respectively at 8 days (Figs. 9c and d).

Discussion
Plants were exposed to short-term salt stress triggers osmotic stress, resulting in reduced water absorption, redox imbalance, stomatal closure, and inhibiting new leaf growth and development of root systems 32 ; however, long-term salinity causes ionic toxicity due to Na + accumulation in adult leaves, caused premature senescence, thus reduced the rate of photosynthesis and nutrient accumulation 33 . Previous studies have shown that excessive salinity significantly inhibited plant growth and biomass accumulation, resulted in reduced plant growth and photosynthetic parameters 9,34 . Rachmawati et al. demonstrated that the reduced root surface area under salt stress lead to reduced water absorption, further inhibited cell division process and cell enlarged, resulting in reduced root length and root biomass 35 .
In the present study, salinity treatment (50 mM NaCl) significantly reduced shoot and root growth morphological indexes of the two rice varieties. Notably, during 8-10 days, salinity treatment increased the root biomass of Chaoyouqianhao, while the root biomass strongly decreased in Huanghuazhan (Figs. 2c and d), which also similar to previous result 36 . However, application of ABA partially or completely reversed the decrease of these indexes, i.e., the plant height, stem diameter, leaf area, root growth (RL, RSA, RV, RAD) and biomass of shoots and roots (Table 1). Similar results was observed in trifoliate orange 9 and wheat 15 . Plant chlorophyll content is widely used as an indicator of the level of abiotic tolerance. Ample evidence showed that plants were exposed to stress environments like salinity resulted in reduced chlorophyll content, resulting in overall growth retardation 37 . In fact, the change of photosynthetic pigment, especially chlorophyll content under salt stress conditions still remained controversial and no clear conclusion has been drawn. It is demonstrated that the increase in chlorophyll content is temporary, mainly due to the positive effect of salinity 38 , At present, we found that salinity treatment exhibited an increase trend in chlorophyll a, chlorophyll b, carotenoid and total chlorophyll contents except at 6 days after salt stress, in Chaoyouqianhao (Fig. 3a, c, e, g), which consistent with the results of Gadelha et al., who demonstrated that the increased chlorophyll a, b and total chlorophyll contents were observed in rice seedlings under salt stress (80 mM NaCl) for 6 days 39 . Meanwhile, the decreased in these photosynthetic pigments in Huanghuazhan may be explained as the enhancement of chlorophyll-lase activity caused by salinity and enable chlorophyll breakdown 40 . It is speculated that the differences in chlorophyll content are related to the cultivar characteristics. On the other hand, ABA + NaCl significantly enhanced the photosynthetic pigments in www.nature.com/scientificreports/ 6-10 days in both of the two rice varieties (Figs. 3a-h). These results implied that ABA reduced the damage of salt stress to the photosynthetic cytochrome protein complex, and thus improved the photosynthetic capacity of rice. Salt stress greatly inhibited biomass accumulation, resulting in reduced leaf area and photosynthetic parameters 34,41 . Hu et al demonstrated that photosynthetic parameters, Qy and Qp significantly decreased in the presence of salinity 9 . A similar result was observed in our study, showing that salt stress reduced Pn, Gs, Ci and Tr in the two rice genotypes (Fig. 4). Notably, the decreased photosynthetic parameters in Huanghuazhan was greater than that in Chaoyouqianhao, which was in harmony with that obtained by previous study 39 . Shibata et al. explained that the reduction in Fv/Fm caused by salinity was due to the photosynthetic electron transfer after light capture not in time, but rather directly consumed light energy via chlorophyll conversion, leading in a  roots (a, b) and leaves (c, d) of two rice cultivar seedlings under salt stress. Values are shown as the mean ± standard and derived from four replicates (n = 4). Bars indicate standard errors. Different letters above the bars indicate significant differences at the five percent significant level according to Duncan's multiple range tests. Table 4. Effects of exogenous ABA on endogenous phytohormone at 8 days after salinity. ACC indicates 1-Aminocyclopropanecarboxylic acid, TZ indicates trans-Zeatin, IPA indicates N6-isopentenyladenosine, IAA indicated Indole-3-acetic acid, ABA indicates ( +)-Abscisic acid. Q 0 treatment of water, Q 1 treatment with ABA, Q 2 treatment with NaCl, Q 3 treatment with ABA + NaCl in Chaoyouqianhao. Z 0 treatment of water, Z 1 treatment with ABA, Z 2 treatment with NaCl, Z 3 treatment with ABA + NaCl in Huanghuazhan. Values are mean ± standard error of three replicates (n = 3). Within each column, different letters indicate significant difference at the five percent significant level according to Duncan's multiple range tests.  42 . In the present study, we found that salinity inhibited Fv/Fm, Fv/Fo, Fm/Fo and Qp at 8 days after salt stress, whereas NPQ significantly increased in rice seedlings especially Huanghuazhan (on average by 118.77%) in 4-6 days (Table 3), the present results were in agreement with that of previous study 43 . The stronger increase in NPQ of Huanghuazhan demonstrated that the absorption and conversion process of chlorophyll were improved to disperse the heat to a greater extent to avoid light damage 44 . These results suggested that salt stress could reduce the biomass of rice shoots and roots via inhibiting photosynthesis and fluorescence processes as well as reducing the amount of photosynthetic pigments.
The salinity-induced damage to rice is mainly caused by Na +33 . Excessive Na + caused ion imbalance (also known as ion poisoning) in plants 45 . Results of Tavakkoli et al. showed that the absorption of nitrogen, phosphorus, calcium and other nutrients reduced under salt stress, and damaged the aboveground tissue function, especially by inhibiting the photosynthesis of chloroplasts 46 . In this study, we made a comprehensive assessment on morphological and physiological attributes in 2-10 days, and we found the optimal attributes were recorded at 8 days after salinity, thus we collected samples at the 8th day for ion analysis. In the present study, salinity stress triggered a large amount of Na + contents while reduced K + contents in roots and leaves of the two rice varieties (Fig. 5). Specially, the large influx of Na + , activating plasma membrane depolarization, in turn activates the K + efflux channels, resulting in K + efflux and increases Na + /K + in the cytoplasm. Similar results were found in a research of melatonin application on rice under salt stress 47 . Notably, the accumulation of Na + contents in leaves was far greater than that in roots, which may be attributed to that Na + can only return to the root via the phloem, although Na + is transported to the shoot via the xylem, suggesting that Na + is strongly unidirectional, leading to the accumulation of Na + in the leaves so that leaves are more susceptible to Na + than the roots 48 . Moreover, Na + contents in leaves of Huanghuazhan were more than that of Chaoyouqianhao, which was in harmony with those obtained by Chuamnakthong et al., who showed that the aboveground part of salt-tolerant rice has a strong Na + efflux capacity as well as a low Na + /K + when compared with salt-sensitive rice 49 . For example, the Na + content accumulated in the leaves of salt-sensitive cv. IR29 was 5 ~ 10 times that of the salt-tolerant rice cultivars BK, FL478, or Pokkali. Similar results were also found in a result of previous research 39 . Both Ca 2+ and Mg 2+ are essential nutrients in the process of plant growth and development and participate in many important physiological regulation processes. For example, Ca 2+ plays an important role in the stability of cell wall, cell membrane, membrane-binding protein, the regulation of inorganic ion transport and the second messenger in cell physiological and biochemical reactions 50 . Mg 2+ is the main mineral element of chlorophyll, which directly affects the photosynthesis of crops, additionally, Mg 2+ could participate in carbon fixation and carbon metabolism as coenzyme factor in plants. The present study showed that Mg 2+ and Ca 2+ contents were inhibited by salinity in leaves of two varieties, whereas Ca 2+ contents significantly increased in the roots of Chaoyouqianhao. It has been shown that the cytosolic Ca 2+ concentration reduced under salt stress conditions, while many studies have shown the enhanced levels of cytosolic Ca 2+ under salt stress 51 . Results suggested that the cytosolic Ca 2+ concentration was not uniform and it could vary with species and type of cells 51 .
Our results suggested that excluding Na + ions, maintaining the low Na + content in the roots and shoots is an important mechanism for plants tolerating high salinity.
Free radicals such as O 2 − , OH − and H 2 O 2 are known to be involved in regulating various signaling cascades associated with multiple biological functions of plants 52 . However, high concentration of free radicals-induced lipid peroxidation has been widely used in ROS to promote cell membrane damage in different stress environments and the MDA levels produced during membrane lipid peroxidation are often used as an indicator of oxidative damage 53 . Enzymatic-antioxidants and non-enzymatic antioxidants are an essential strategy against the environmental stresses. The former enzymatic-antioxidants including SOD, POD, CAT and APX, and the latter such as AsA and GSH directly involved in ROS homeostasis and detoxification in plant cells 5 . In the present study, salinity stress triggered an increase of MDA in roots and leaves of Chaoyouqianhao, while the greater increased MDA in roots and leaves of Huanghuazhan were observed towards the changed treatment time (Fig. 6); this results implied that salt stress causes a higher degree of peroxidative damage to the leaf and root tissues in Huanghuazhan than that in Chaoyouqianhao. Meanwhile, the contents of MDA in leaves were more than that in roots suggested that salinity stress caused a greater damage to leaves than to roots. H 2 O 2 is widely involved in plant metabolism (e. g., cell wall biosynthesis) and plays central roles in many plant signaling pathways, including stress sensing, photosynthesis modulation, pathogen response, programmed cell death, hormonal action, and plant growth and development [54][55][56] . However, H 2 O 2 can also be converted into harmful hydroxyl radicals by the fenton-type reaction, and then exerts multiple toxic effects in different cellular compartments 57 . Our study showed that salinity toxicity triggered increased H 2 O 2 content in roots of Chaoyouqianhao and Huanghuazhan. Notably, the decreased H 2 O 2 content due to salinity toxicity was observed in leaves of Huanghuazhan, similar results were obtained in barley 58 . Even so, Ye et al. showed that low H 2 O 2 content was observed in salt-tolerant varieties 59 . Sadak declared that H 2 O 2 played a key role in the improvement of salt stress by enhancing multiple defense-related mechanisms, with H 2 O 2 molecules penetrating through the plasma membrane into different cellular compartments to mitigate the deleterious effects of salinity as a central role in activating various signal transduction pathways 60 . Furthermore, different studies have also demonstrated its involvement in triggering various genes, transcription factors, and phytohormones associated with salt stress tolerance 61,62 . All of these explained for the reason of the greater H 2 O 2 in leaves of Chaoyouqianhao than that of Huanghuazhan in our study, as similarly reported by previous study 63 .
Previous studies have demonstrated that salinity stress activated the antioxidant systems and conferred salt tolerance [64][65][66] . Nevertheless, effects of salt stress on plant antioxidant systems are controversial. Qiu et al. showed that the activity of SOD, POD, CAT, APX decreased and the contents of GSH and AsA increased under salt stress in wheat seedlings 66 . Altaf et al. demonstrated that salt stress significantly increased SOD, CAT, APX activity and the level of GSH and AsA in tomato seedlings 65 . Our study showed that salinity increased the level of CAT, APX, POD and SOD in roots and leaves, particularly at 6 days or 8 days (Fig. 7). Interestingly, the increased Scientific Reports | (2022) 12:8228 | https://doi.org/10.1038/s41598-022-11408-0 www.nature.com/scientificreports/ CAT, APX and POD activity in leaves were more prominently than that in roots imposition with salinity. Similar results were observed in lentil seedlings 28 . This results demonstrated that the higher antioxidant enzyme activity in leaves could eliminate ROS and MDA so that reduced membrane damage when compared with root, which is also corresponded to the accumulation of more MDA and Na+ content in leaves. Besides, salinity increased stronger POD activity in roots, which similar to previous study of Meneguzzo et al., who demonstrated that APX activity was significantly higher in the root tissue than in the shoots of wheat seedlings 67 . Notably, the slight increased SOD in roots and leaves were observed at the early stage of salt stress, which in line with many previous studies 68,69 . This phenomenon may be due to that a non-enzymatic pathway was used to convert O 2 − to H 2 O 2 using antioxidants such as GSH and AsA. Last but not least, we found that lower H 2 O 2 and MDA contents in leaves were maintain in Huanghuazhan at the late stage of salt stress (Figs. 6f and h), which was corresponded to the lower SOD activity (Fig. 7r), indicated that the higher activities of CAT, APX and POD in leaves participated in the decomposition of H 2 O 2 , thus reducing the H 2 O 2 level and membrane damage.
The AsA-GSH cycle is considered as one of the most important nonenzymatic antioxidant systems 70 . The present study of enhancement of AsA and GSH contents in roots corroborated with the findings of previous research 71 . It was of note that AsA and GSH contents in leaves of the two rice genotypes inclined to reduce at the late stage of salt stress. As the most abundant antioxidant in plants, AsA is widely involved in the elimination of ROS during stress response so that protect the major macromolecules from oxidative damage 72 . Thus, this may explained that a greater oxidative damage to leaves than to roots. On the other hand, the decreased GSH may be attributed to the elimination of ROS in plant cells by depletion of GSH.
Plant hormones play a crucial role in plant growth and development. To mitigate salt stress at different developmental stages, the levels of plant hormones such as ABA, IAA, GA, JA, BR, SA and ethylene were modulated the physiological response of plants under salt stress 73 . In this study, we collected samples at the 8th day for endogenous hormones analysis. Hu et al demonstrated that salt stress significantly reduced IAA content and significantly increased zeatin riboside 9 , similar to our study, showing that ACC, IPA (a cytokinin of bound state), TZ (a natural plant cytokinin) and IAA reduced under salinity in Huanghuazhan. Lin and Kao demonstrated that there was no increase in ABA content in the roots under salinity stress, suggested that the inhibition of root growth in rice seedlings is unlikely to be mediated by ABA, however, this does not mean that the amount of ABA does not increase under salt stress; exactly, it may be due to that the ABA transfers from the roots to the shoots 74 . Conversely, our results showed that salt stress showed a greater increment in ACC, IPA, IAA and ABA in leaves of Chaoyouqianhao, whereas only the increased ABA content was observed in Huanghuazhan. Similar results were obtained by Zahedi et al., who reported that IAA and ABA contents increased in the presence of salinity in olive seedlings 75 .
ABA regulates the water state of plants by guarding cells, which contributes to overcome the adverse effects of salinity on growth, photosynthesis, and assimilates transport 3 . Foliar spraying ABA induced the stomatal closure and reduced the transpiration rate, which could be used as an anti-transpiration agent in production 76 . In the present study, ABA application strongly enhanced the photosynthetic parameters Pn, Gs and Tr in presence of salinity, it may be driven by an increased Gs, enhanced stomatal reopen and improved its function, thus improve Tr and maintain the stable state of Pn under salt stress; on the other hand, it may be that ABA effectively improves chlorophyll content and promotes the potential activity of PS II (Fv/Fo), thus enhancing plant photosynthesis 77 . Similar results were observed in lettuce 78 . Additionally, application of ABA stimulates the antioxidant system to protect salt toxicity from exaggerated photosynthetic electron transport in plants to enhance carbon assimilation 79 .
One of the mechanisms underlying plant salinity tolerance is the selective absorption and accumulation of inorganic ions, mainly Na + , K + , and Cl −80 . It was demonstrated that ABA was a high efficient plant growth regulator in decreasing Na + and Cl − contents and Na + /K + ratio, increasing K + and Ca 2+ concentrations 81 . In the present study, ABA significantly reduced Na + contents in leaves of the two rice cultivars as well as in roots of Chaoyouqianhao, implied that ABA reduced transpiration by inhibiting K + efflux channel, kept stomatal closure and resulted in a reduction in ion absorption in rices, thus a reduction in Na + absorption was observed. On the other hand, Ca 2+ as a secondary messenger initiates the stress signal transduction, leading to salinity adaptation. Previous study have showed that exogenous calcium reduced the effect of NaCl presumably by facilitating higher K + /Na + selectivity 82 , which was well in line with our study, showing that a significant increase in Ca 2+ in roots and leaves. In addition, it was found that Mg 2+ increased under ABA-mediated salt stress, which may contribute to the enhancement of vacuole growth in leaves and roots, thereby isolating Na + into vacuoles and limiting the accumulation of Na + in shoot 83 .
Many studies have demonstrated that ABA could increase the antioxidant enzymes activity and alleviate the stress-triggered oxidative damage to plants 15,21,84 . However, the data that support this hypothesis remain inconsistent. For example, application of ABA in kiwi fruit under drought stress significantly improved the activity of POD, CAT, SOD, and APX 70 , whereas some results shown that ABA decreased CAT activity but significantly increased the activity of SOD and POD 85 . The present study displayed that ABA partly or completely increased the activity of CAT, APX, POD and SOD in roots, which depended on salt treatment time, suggesting that exogenous ABA upregulated antioxidant enzyme activity, thereby eliminating the ROS over-accumulation and protecting the cell membrane from oxidative damage. Conversely, these enzymes exhibited decreased trend in leaves of Huanghuazhan at 6 or 8 days after salt stress mediated by ABA. It may be that SOD is involved in elimination a part of ROS, resulting in reduced ROS generation and with that CAT,APX,POD activity decreased, which may also be due to the irreversible damage of plants under salt stress, caused the structure of enzyme protein damaged and resulting in decreased the enzyme activity. As a key enzyme of phenylpropanoids metabolism in plants, PAL plays an important role in abiotic stress. The increased activity under ABA treatment is presumably due to the up-regulation of PAL gene expression, thus improve the ability of stress resistance and promote the www.nature.com/scientificreports/ seedlings to adapt to the salt stress environment. However, the regulation of PAL gene in ABA-mediated salt stress pathway needs further study. Previous study showed that exogenous application of GSH and AsA lead to reduced ROS accumulation and increased salt tolerance 86,87 . In our study, ABA strongly improved the level of AsA and PAL in the two rice seedlings, particularly in roots. As a key enzyme of phenylpropanoids metabolism in plants, PAL plays an important role in abiotic stress. The increased activity under ABA treatment is presumably due to the upregulation of PAL gene expression, which regulates the synthesis and accumulation of flavonoid 88 , thus improve the ability of stress resistance and promote the seedlings to adapt to the salt stress environment. However, the regulation of PAL gene in ABA-mediated salt stress pathway needs further study. Notably, our results showed that ABA significantly reduced the level of GSH in roots at 6 or 8 days after salt stress in the two rice varieties, which consistent with previous reports 9, 16 . Liting et al. pointed out that the differences of AsA and GSH in root and leaf tissues may be related to different functions, growth environment, and sensitivity of roots and leaves to drought stress stimulation and ABA 14 . Thus, we implied that the mechanism of ABA is different in the root and leaf tissues of rice seedlings.
Plant hormones are key endogenous chemical signals that plants coordinate plant growth and development under both optimal conditions and environmental stress 89 . Liu et al. pointed out that ABA significantly increased endogenous hormone contents such as ABA, GA, IAA and ZT 30 . Similar to our study, showing that ABA application increased the contents of ACC, TZ, IPA, IAA and ABA in Chaoyouqianhao (Table 4). Particularly, ACC content exhibited a strong increase (212.00%) when compared with salinity treatment; notably, it showed a stronger increase (317.80%) in Huanghuazhan. As a direct biosynthetic precursors of ethylene, ACC play an equal essential role in response to salt tolerance in rice, which has been extensively studied. Namely, promoting ethylene biosynthesis and signaling transduction can improve salt tolerance in plants 90 . Moreover, CTK belong to a class of plant growth substances (plant hormones) which includes IPA and TZ, regulating diverse events in plant growth and development. Results in our study showed that exogenous ABA increased the level of IPA and TZ under salt stress, especially in Huanghuazhan, which contributes to synthesis more CTK and thus regulated salt stress tolerance. Last but not least, the increased ABA contents mediated by ABA-stimulated under salt stress further regulated ROS scavenging, ion homeostasis, and stomatal closure 91 .
In conclusion, the present study reveals the physiological mechanisms underlying ABA-mediated the mitigation of salinity stress to plants. We focused on the response of root and leaf tissues to salt stress in two different rice cultivars and the alleviation effect of ABA application on salinity toxicity. Salt stress caused a significant reduction in plant morphological indexes and great changes in physiological characteristic of two rice cultivars. Foliar application of ABA mitigated the inhibitory effect caused by salinity on plant growth by improving photosynthesis, modifying the antioxidant enzymes (CAT, APX, POD, PAL) and non-antioxidant enzymes (AsA and GSH) as well as reducing oxidative damage, ultimately maintenance of ionic (low Na + and high K + /Na + ) balance and salinity tolerance. Our study simultaneously demonstrated that the beneficial response and adaptation to salt stress requires the integration and coordination of multiple phytohormones. Further studies are needed to investigate the molecular mechanisms of ABA-mediated mitigation of salinity toxicity to plants. (A pattern diagram (Fig. 10) was added in Attachments).

Materials and methods
Plant materials and growth conditions. Seeds of two rice (Oryza Sativa L.) varieties, one inbred rice 'Huanghuazhan' (Z) and the other hybrid rice 'Chaoyouqianhao' (Q) were obtained from Guangdong Tianhong Seed Company Limited, Zhanjiang. Seeds were surface sterilized with a 2.5% sodium hypochlorite solution for 15 min, followed by rinsing three times with distilled water, and then the seeds were germinated with distilled water in the dark for 2 days at 30 ℃. After that 75 uniformly germinated seeds were used as experimental materials for growing per plastic pot (19.5 × 14.5 × 17.5 cm) containing 3.0 kg of substrates (Latosol: sand = 3:1, v/v). In the present test, a factorial experiment based on completely randomized design was carried out. Briefly, 30 pots were used per plot (represent one of eight treatments, treatments were list as following), and therefore a total of 240 pots were used in the study.
When the rice seedlings had naturally grown to the 2 leaf/1 heart stage (8 days after planting), seedlings were foliar spraying with 5 mg L −1 S-ABA (provided by Sichuan Lomon Fusheng Technology Co., Ltd), using a small hand-held sprayer, each pot was added with 20 mL liquid. Specially, the concentration of ABA was chosen according to previous pot experiment study (Fig. 1), then 25 mM NaCl (w/w) solution was added to each pot after 24 h, and the other 25 mM NaCl solution was added on the 3 rd day of ABA treatment to got the desired salinity level (50 mM). The following applications of S-ABA and NaCl were applied to each experimental pot: (i) (Q0 or Z0): 0 mg L −1 S-ABA + 0 mM NaCl, (ii) (Q1 or Z1): 5 mg L −1 S-ABA + 0 mM NaCl, (iii) (Q2 or Z2): 0 mg L −1 S-ABA + 50 mM NaCl, (iv) (Q3 or Z3): 5 mg L −1 S-ABA + 50 mM NaCl. The plant samples were harvested at 2, 4, 6, 8, 10 days after salt stress for morphological and physiological analysis.
Observations. Growth response measurements. Samples of roots and shoots were collected at the treatment intervals. Plant height, stem base width, leaf area, leaf length, shoot dry weight and root dry weight were measured. For determining dry weight, shoots and roots were separately placed in paper bags, and then placed in an oven at 80 ℃ for 48 h. For determining RL, RSA, RV and RAD, root system scanner (Epson Perfection V800 Photo (Epson Indonesia Inc.)) and analysis software WinRHIZO were used. All the values for each parameter were mean of at least four independent replicates. Root to shoot ratio was calculated according to the following formula: Root to shoot ratio = Root dry weight/Shoot dry weight Measurement of photosynthetic parameters. After 2, 4, 6, 8 and 10 days of salt stress, P n , G s , C i and T r were measured by using portable photosynthesis system LI-6400 (LI-COR, Inc., USA). The measurements were made between 14:00 and 16:00 P.M. The CO 2 concentration in the leaf chamber was 400 μmol·mol −1 , the air velocity was 500 μmol·s −1 , the light intensity was 1000 μmol·m −2 ·s −1 , the leaf temperature was 32 ± 1℃, and the relative air humidity was between 70 and 80%. Measurement of ions contents. After 8 days of salt stress, Na + , K + , Mg 2+ and Ca 2+ contents were determined in 150 mg dry mass (DW) of leaves and roots. Samples were digested in 6 ml of HNO 3 /HClO 4 (4:1, v/v), and supernatants after clarifying the digestate were analyzed. Na + , Mg 2+ and Ca 2+ contents were measured by Inductively Coupled Plasma Emission Spectrometry (Prodigy XP, LEEMAN, Inc., USA) and the K + content was measured by flame photometer (Sherwood M410, Inc., UK). www.nature.com/scientificreports/ Analysis of lipid peroxidation. Lipid peroxidation was measured in terms of MDA level using the TBA method as described by Cakmak and Horst 93 . Absorbance of the supernatant was measured at 450, 532 and 600 nm. The MDA-TBA complex was quantified using the extinction coefficient as 155 mM −1 cm −1 . The determination of H 2 O 2 concentration was measured according to previous study 94 .

Measurement
Measurement of antioxidant enzyme activity. 0.5 g of fresh root and leaf tissues were ground into fine powder under liquid nitrogen, and then 10 mL of precooled phosphate buffer (50 mM, pH 7.8) containing 1.0% (w/v) PVP was added and homogenized. Mixture was centrifuged for 40 min at 7000 × g and 4 ℃. The obtained supernatant was used for enzyme assays as the crude enzyme preparation. The CAT 95 , APX 96 and POD 97 activity were assayed as described by Ekinci et al., Nakano and Asada, Chance and Machly, respectively. Since the addition of H 2 O 2 , the absorbance changes were monitored for 120 s at 240 nm, 290 nm and 470 nm for assayed the CAT, APX and POD activities, respectively. The SOD activity was assayed as described by Spychalla and Desborough 98 . One unit of SOD activity was defined as the amount of enzyme needed to cause 50% inhibition of the photoreduction of NBT as monitored at 560 nm.
Measurement of antioxidant molecules. The AsA content was measured according to the method of Kampfenkel 99 . The absorbance value was read at 534 nm. The level of GSH content was assayed as described by Tyburski