The beneficial effect that ALA improves plant salt tolerance has been received much attention in recent years [7, 24], but the regulatory mechanisms remain unclear. In most of previous studies, the increases of antioxidant enzyme activities are considered as the main mechanisms for ALA to improve salt tolerance [8, 23, 43, 44, 45]. Additionally, leaf pigments and photosynthesis [19, 46], proline accumulation [47], especially ion homeostasis [48] are also considered important. A great amount of salt ions is absorbed by plant roots, transported to shoots and accumulated in leaves, which is the lethal reason for salt injury and plant death. How to cope with harmful ions to maintain ion homeostasis under salt stress should be the key for plant salt tolerance. No clear relationship between ALA-induced salt tolerance and ion distribution has been known [49] until a recent report of our group. We found that ALA induced H2O2 accumulation in strawberry roots to selectively retain Na+ in the underground with less accumulation in leaves under salt stress. We estimated Na+ and K+ levels in different tissues and xylem sap with several methods. All results turned out that NaCl stress significantly increased the Na+ content in both leaves and roots with higher Na+/K+, and ALA further improved the root Na+ content but depressed it in leaves [25]. Thus, ALA-induced Na+/K+ ratio increased greatly in roots but decreased in leaves. In xylem sap, ALA induced 33% decrease of Na+ concentration but K+ concentration was not different from the NaCl treatment without ALA. This means that ALA induces Na+ selective retention in roots (rather than K+ selective transport upward) with less transport upward to shoots. The root intercepted Na+ may be extruded by plasmolemma-located Na+/H+ antiporter (encoded by SOS1) into soil solution, or sequestrated into vacuoles by tonoplast-located Na+/H+ antiporter (encoded by NHX1) to store as cheap osmotic solutes. Additionally, HKT1, coding a Na+-selective transporter was also induced up-expression by ALA, which is responsible for Na+ unloading from xylem vessels to parenchyma cells in roots to reduce ion concentrations in xylem sap [1], or Na+ removal from xylem sap to phloem through plasmodesmata via symplastic diffusion, then downward to roots, and avoiding too much Na+ accumulation in shoots [50]. Thus, the findings, different from the other previous reports, open a new insight for us to understand the mechanisms of ALA in inducing salt tolerance, especially ion homeostasis strategy [26].
In present work, we once again observed that ALA treatment decreased the leaf Na+ content but significantly increased the retention in the roots of strawberry (Fig. 6A). It is quite agreed with our previous findings [25]. Furthermore, we also found Cl-, like Na+, was preferably retained in roots with less accumulation in leaves after ALA treatment (Fig. 6B). In a transgenic canola (Brassica napus) which can over-produce endogenous ALA, the leaf Cl- content is strictly limited to a very low level, even seedlings were stressed by 450 mM NaCl [51]. Additionally, in the previous study when the ion levels of xylem sap was measured, it was also found that ALA significantly depressed [Cl-]xylem under salt stress, which were 2.39, 6.20 and 3.50 μmol L-1 in the control, NaCl stress and NaCl + ALA, respectively. Thus, ALA induces plants to intercept both Na+ and Cl- in roots to decrease the toxic ion contents in the shoots. The opinion is agreed with Hanin et al. [52], who pointed out that plants have evolved mainly two types of tolerance mechanisms to cope with salt stress, one is limiting the entry of salt by the roots, and the second is controlling its concentration and distribution. Comparatively, the other responses in ALA-treated plants, such as higher levels of leaf chlorophylls and photosynthetic capacity may be secondary mechanisms for plants salt tolerance [46, 47]. Additionally, we reported that ALA increased the K+ content in both leaves and roots, but the K+ concentration in xylem sap of strawberry was not improved by ALA treatment under salt stress [25]. Thus, K+ level is not the most critical when ALA improves salt tolerance of strawberry.
In the previous report [25], the effect of ALA-induced root Na+ retention was ascribed to the role of H2O2 on up-expressions of Na+ transporter genes. H2O2 is known as a reaction oxygen species (ROS), as well as an important cellular signal. However, in most studies about ALA, H2O2 was considered as ROS rather than a cellular signal [23]. Never attention had been payed to different responses of H2O2 between shoots and roots [7] until Wu et al., who found that salt stress induced H2O2 increase in both leaves and roots, and ALA induced more H2O2 increase in roots but depressed in leaves [25]. The tissue-specific response is confirmed recently [53] and observed in present work once again (Fig. 4), which may be an important characteristic for ALA to induce plant stress tolerance. In another report, it was found that ALA decreased the O2•ˉ production rates in both leaves and roots strawberry under salt stress, however, there was no H2O2 information available [56]. In present work, we do not only validate the results of Wu et al. [25], but reveal that SNP, a NO donor also improves H2O2 increase in roots of strawberry, while Na2WO4 or cPTIO inhibit the H2O2 increase induced by ALA (Fig. 4). This means that ALA-induced H2O2 increase in roots of strawberry is dependent on NO presence. In another word, NO may be a signal located at the upstream of H2O2, involved in ALA-induced plant salt tolerance. However, in cucumber roots, ALA did not induce H2O2 increase [54]. The reason for this difference needs further clarification.
It is the first time to show NO involved in the signal route that ALA improves salt tolerance of strawberry (Fig. 1). NO is an important gaseous signal molecule involved in regulation of plant response to salt stress [33, 38]. In higher plants, nitrate reductase (NR) is the key enzyme for NO production [29, 55]. Since the activity is dependent on molybdenum, Na2WO4 is often used as competitive inhibitor [57]. In pakchoi [58] or barley [59], ALA has been found to induce NR up-expression and enzyme activity. In present work, we observed that NaCl and ALA significantly induced NR up-expression in roots of strawberry, while Na2WO4 completely eliminated the expression induced by ALA (Fig.2B). Thus, the inhibition of NR expression by Na2WO4 is responsible for the block of NO generation in strawberry under salt stress. It is interesting to notice that ALA induces NO accumulation in roots of strawberry but without effect in leaves (Fig. 2A), suggesting that NO generation induced by ALA is also tissue-specific, similar with H2O2 (Fig. 4). We used DAF-FM DA, a highly specific NO fluorescent probe to stain root tips of strawberry, the visual results are agreed with measurements with spectrophotometer (Fig. 3). Thus, both salinity and ALA greatly induce NO accumulation in roots of strawberry. However, the reason for ALA to induce NO accumulation only in roots not in leaves is not clear now. Yet, our unpublished data shows that carbon monoxide (CO), released by catalysis of heme oxygenase may be involved in the ALA-NO-H2O2 signaling route in ALA regulating salt tolerance of strawberry. We have found that hematin, a CO donor can induce both increases of NO and H2O2 levels in the roots of strawberry under salt stress, while hemoglobin, a CO scavenger can eliminate the root Na+ interception induced by ALA. ALA is the key precursor of heme biosynthesis. Significant higher levels of endogenous heme have been reported in the Yhem1 transgenic Arabidopsis [60] or the exogenous ALA treated pakchoi seedlings [21]. In mouse macrophage cell lines, exogenous ALA enhances the heme oxygenase gene (HO-1) expression, which catalyzes the rate-limiting step in the oxidative degradation of heme to free iron, biliverdin and CO [61]. Whether the similar mechanism occurs in higher plants is not known, but we can deduce that the signals of ALA in improving salt tolerance may exist in its conversion into porphyrin compounds and their metabolites, such as CO and NO.
However, SNP is an exogenous substance, which may generate cyanide and ferricyanide ions beside NO in aqueous solution [62]. One might argue that CN- or Fe(CN)63- instead of NO is the key active factor in salt tolerance improvement induced by SNP. Fortunately, in an independent experiment, we observed that 5 μM cPTIO completely eliminated the effect of SNP on promotion of salt tolerance of strawberry, while treatments with 10 - 100 μM K4Fe(CN)6 did not exhibit significant effect on salt tolerance (Fig. 1S). Therefore, the promotion of SNP on salt tolerance can only be attributed to the effect of NO.
NO has been reported to induce antioxidant enzyme activity under salt stress [39, 63]. In present work, we found that salt stress, ALA and SNP all induced increases of antioxidant enzyme activity in strawberry, but eliminated by Na2WO4 or cPTIO (Fig. 5). These mean that ALA-induced antioxidant enzyme activity is dependent on NO presence. However, no tissue-specific responses of antioxidant enzyme activity to ALA are found here. Thus, the response of H2O2 accumulation to ALA is different from the antioxidant enzyme activity. In fact, the activities of all three enzymes measured in the study changed coincidently between leaves and roots after treatments. We can also see that leaves possess higher activity of SOD but lower POD than roots, and the CAT activities are comparable between roots and leaves. However, the biological meaning of differences is not clear. Correlation analysis shows that the root H2O2 content was significantly positive correlated with the antioxidant enzyme activities in roots, where rSOD = 0.898*, rPOD = 0.944**, rCAT = 0.936**, respectively. However, the leaf H2O2 content does not correlate with three enzyme activities (P > 0.05). It seems that the relations between H2O2 and antioxidant enzyme activities in the leaves are more complex than that in the roots, which needs study further.
It is known that the most important genes related with Na+ transport are NHX1, HKT1, and SOSs in Arabidopsis [64, 65]. In the previous report, Wu et al. proposed that ALA-induced H2O2 was necessary for up-expression of these genes (including FaNHX1, FaHKT1 and FaSOS1) when strawberry was subject to salt stress [25]. In present study, both ALA and SNP enhanced expressions of FaSOSs (Fig. 6A), FaNHX1 and FaHKT1 (Fig. 6B) significantly in strawberry roots, while Na2WO4 or cPTIO eliminated the effect induced by ALA. Thus, we propose that NO is another necessary component in the signal cascade of ALA-induced salt tolerance of strawberry, at the up-stream of H2O2, responsible for up-regulation of Na+ transporter gene expressions. In Jatropha curcas, NO has been suggested to decrease harmful ion accumulation under salt stress [38]. ALA-induced salt tolerance may accord with the similar signal route.
In SOS family of Arabidopsis, six genes have been identified. All of them consist of an SOS signaling route responsible for Na+ transport [66]. AtSOS1 is the Na+/H+ antiporter located in plasma membrane. AtSOS2 interacts with AtSOS3 to form SOS2/SOS3 complex, in turn phosphorylating and activating AtSOS1 [67]. AtSOS4 is a pyridoxal kinase (PLase) involved in the biosynthesis of pyridoxal-5-phophate (PLP), regulating Na+, K+ channel or transporter. Thus, sos4 mutant is hypersensitive to KCl and NaCl [68]. In strawberry, we find out four genes of FaSOS family, where FaSOS1 and FaSOS2 can be coincidently up-regulated by ALA and SNP under salt stress, but the effect is eliminated by Na2WO4 or cPTIO (Fig. 7A). These suggest that ALA as well as its induced NO is sufficient and necessary for the gene expressions. For FaSOS3, ALA can induce its up-expression, but cPTIO cannot eliminate the effect. This may imply that NO is sufficient but not necessary. There may be other regulatory branches. For FaSOS4, ALA rather than SNP can up-regulate its expression, and two inhibitors eliminate the promotion. This may suggest that NO is necessary but not sufficient. Obviously, the regulatory mechanisms of FaSOSs are more complex than we have known. Beside NO, other factors such as ABI2, 14-3-3 [66], WRKY40 [67], ethylene signals [69] may also be involved in regulation of SOS signal route. Furthermore, SOS2 and SOS3 complex can interact with CIPK and CBL proteins [4], which is an important node linking H2O2 and salt stress [70]. From our results here, it seems that the synergy of gene up-expressions by ALA under salt stress causes Na+ extruded from the cytosol out of cells, where NO signaling is necessary in strawberry roots.
CLCs are a gene family coding important anion channels or transporters, widely distributed on the membranes of prokaryotic and eukaryotic cells to mediate Cl- or NO3- transport [71]. In Arabidopsis, there are seven members of CLCs, including AtCLC-a ~ AtCLC-g [72]. The function and subcellular location of AtCLCs have been identified. For example, AtCLC-a as well as the highly homologues AtCLC-b, is responsible for NO3-/H+ or Cl-/H+ transport across tonoplast [73]. AtCLC-c is also located on tonoplast, mainly in guard cells, may be involved in stomatal regulation and beneficial for plant salt tolerance [74]. AtCLC-d is localized to the membrane of Golgi bodies. AtCLCe is targeted to the thylakoid membrane in chloroplast and related to photosynthesis activity. AtCLC-f is localized on the Golgi apparatus and mainly responsible for Cl- transport [75] while AtCLC-g mainly distributed in leaf mesophyll and phloem cells [76]. Based on the sequences of Arabidopsis, we obtained part of the respective homologs in strawberry. Analysis with qRT-PCR shows that the FaCLC expressions were significantly induced by salinity, while doubled or redoubled by ALA (Fig. 6C). Similarly, SNP also induced these gene up-expressions, suggesting that ALA-induced NO is involved in Cl- compartmentation and transport in strawberry. Nevertheless, NR inhibitor Na2WO4 or NO scavenger cPTIO eliminated the up-expression of FaCLC-a, FaCLC-d and FaCLC-f, implying that the gene expressions induced by ALA may be dependent on NO presence. However, the expression of FaCLC-g induced by ALA cannot be blocked by the inhibitors, suggesting its regulation maybe be specific. Anyway, it is interesting to notice that all the gene up-expressions induced by ALA and NO seem to be beneficial for Cl- subcellular compartmentation. It can also be used to reasonably explain the possible mechanisms for ALA to promote Cl- retention in roots (Fig. 5B) and relieve salt injury of strawberries (Fig. 1) when they are subject to salt stress.