5-Aminolevulinic Acid-induced Salt Tolerance in Strawberry: Possible Role of Nitric Oxide on Interception of Salty Ions in Roots

Background: 5-Aminolevunic acid (ALA), as a natural non-protein amino acid and the �rst essential precursor of tetrapyrrole biosynthesis in all living bodies, has been suggested to improve salt tolerance of plants. In the previous work, we reported that ALA induces H 2 O 2 accumulation in roots of strawberry, which is involved in up-regulating Na + transporter gene expressions to intercept Na + in roots with less upward transport. However, the signal route is not clear. Results: In this study, we propose that nitric oxide (NO) is involved in ALA signaling cascade. Therefore, we applied sodium nitrosylpentacy (SNP, NO donor), Na 2 WO 4 (NO biosynthetic inhibitor), and 2, 4-carboxyphenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, NO scavenger) to the culture solution when strawberry (Fragaria × ananassa Duch. cv. ‘Benihoppe’) was stressed by 100 mmol L -1 NaCl with or without exogenous ALA. The results reveal that salinity greatly impaired plant growth while 10 mg L -1 ALA or 10 µM SNP ameliorated the inhibition. When 5 µM Na 2 WO 4 or cPTIO was co-treated, the ALA-improved salt tolerance was almost completely eliminated. This suggests that ALA-improved salt tolerance is dependent on NO presence. We found that salinity caused NO, H 2 O 2 , Na + and Cl - increases in the whole plants, while ALA induced additional increases in roots but signi�cant depressions in leaves. These tissue-specic responses to ALA are important for plant salt tolerance. Conclusion: We propose that the regulation of ALA in roots is critical, which is mediated through NO and then H 2 O 2 signal to up-express genes related with Na + and Cl - transport, selectively retaining Na + and Cl - in roots with less upward transport. The hypothesis can reasonably explain how ALA-treated plants cope with toxic ions under salinity.


Background
Soil salinization is a major problem threatening agricultural production, which seriously inhibits growth and development of crops and results in a decline in yield and quality.Yet, many plants have developed the ability to sense both osmotic stress and ionic stress [1].It has been found that plants subjected to excess NaCl accumulate high Na + concentrations in root tissue within the rst 2 min [2], whereas Na + e ux from the root tissue starts at 10 min [3].These results suggest that excess Na + is rapidly sensed by plants, which triggers the downstream of salt stress responses [4].Reactive oxygen species (ROS) can be induced by salt or osmotic stress to be a species of toxic substances on one hand and, which may be linked to cellular signaling to involve biological regulation on the other hand [5].Na + and K + transporters play key roles in resistant mechanisms to salt stress [1].Mechanisms to reduce cytoplasmic Na + include restricting Na + uptake, increasing Na + e ux, and compartmenta-lizing Na + in the vacuole [4].There are three major transporters that are involved in these physiological processes: tonoplast-localized NHX1, segregating Na + within the vacuole; plasma membrane-localized SOS1 (Na + /H + antiporters), exporting Na + out of the cell; HKT1, removing Na + from the xylem sap into the surrounding xylem parenchyma cells to control long distance root-to-shoot Na + partitioning.They are composed of the most important basis for plants to maintain ion homeostasis under salt stress.5-Aminolevulinic acid (ALA), a new plant growth regulator, is a common precursor of all porphyrin compounds [6].It has been known to regulate plant growth and development [7,8], such as callus induction, adventitious root and bud formation [9], stomatal opening [10][11][12], ower thinning [13], fruit coloration [14][15][16], and stress tolerance [17][18][19].In salt tolerance, ALA can promote seed germination under salt stress [20,21].It also improves leaf photosynthesis under salt stress [22].It has been suggested that ALA alleviates salt stress by increasing antioxidant enzyme activity and reducing membrane injury [23].However, ALA may also modulate plant salt tolerance through its regulating the metabolism of tetrapyrrole and proline accumulation in Brassica napus seedlings [24].Recently, we demonstrated that ALA improved strawberry salt tolerance by stimulating H 2 O 2 level in roots, which in turn regulated the up-expression of genes encoding the ion transmembrane transporters, such as NHX1, SOS1 and HKT1, and then Na + is selectively intercepted in the roots with less upward transport and avoiding ion accumulation in shoots [25].It is a completely new opinion about ALA dealing with plant salt tolerance [26].However, the signaling route from ALA to H 2 O 2 generation is not clear.
It is well known that nitric oxide (NO) as a multifunctional cellular signal, plays an important role in a variety of physiological processes [27].In animals, NO production is predominantly catalyzed by nitric oxide synthases (NOS) [28].In plants, nitrate reductase (NR) is the key enzyme for NO production, which catalyzes nitrite reduction to NO using NAD(P)H as co-factor [29].Up to now, NO has been known to be involved in stomatal movement [30], adventitious root formation [31], avonoid biosynthesis [32], and alleviation of various abiotic stresses [33], such as drought [34,35], chilling [36], freezing [37], and salinity [38].In rice plants, NO induces antioxidant enzyme activity against salt stress [39].In tomato, NO increases photosynthetic rate and chlorophyll uorescence parameters under salt stress [40].Additionally, NO is reported to promote H 2 O 2 accumulation in callus of Populus euphratica, which improves salt tolerance [41].Addition of NO can increase the expression of SOS1 and NHX1 in Avicennia marina under salt stress [42].These results indicate that both NO and ALA improve salt tolerance of plants by promoting H 2 O 2 production and increasing the expression of salt tolerant genes, but the relationship between them is little known.Therefore, the possible role of NO on the salt tolerance of strawberry induced by ALA is studied in present work to understand the possible mechanisms of ALA in alleviating salt injury.

Alleviations of ALA and SNP on plant growth inhibition under salt stress
The dry weights of both shoot and root of strawberry treated with 100 mM NaCl for 9 days were signi cantly lower than that of the control (P < 0. 01), where the root dry weight of salt-treated plant was only 45% of that of the control, while the shoot was 64% of the control (P < 0.01) (Fig. 1).This suggests that salt stress inhibited root growth more than leaf.If plants were treated with ALA, the dry weights of both leaves and roots between NaCl + ALA and the control were not signi cantly different (P > 0.05), suggesting that ALA almost completely eliminated growth inhibition caused by salinity.Speci cally, the dry weight of roots treated with 10 mg L -1 ALA was 79% of the control, while that of shoot was 101%, compared with respective of the control.These indicate that ALA has a strong protective effect on the root growth against salt stress, and it can protect shoot growth avoiding salt injury.Similarly, 10 μM SNP treatment also had alleviating effect on growth inhibition of salt stress, where the dry weight of roots was 70% of the non-salt control, signi cantly higher than that of NaCl alone (P < 0.05).And, the aboveground part of SNP treatment was 94% of the non-salt control (P > 0.05).These suggest that exogenous NO also improves salt tolerance of strawberry, in a model similar with ALA.When NO production inhibitor, sodium tungstate Na 2 WO 4 , or NO scavenger cPTIO, were added with ALA, the effect of ALA to relieve salt inhibition disappeared.The results suggest that the effect of ALA on enhancing salt tolerance of strawberry may depend on the presence of NO.

Effect of ALA and SNP on NO levels in different tissues of strawberry under salt stress
The leaf NO content was generally much higher than that of roots (Fig. 2A).Salt stress induced its doubled increase in both of leaves and roots, and ALA further improved the increase in roots but completely blocked in leaves.Compared with the control, NaCl induced root NO increase by 1.39 times, while ALA induced increase by 3.56 times.However, the leaf NO content in ALA treatment was almost the same with the non-saline control.This exhibits different NO responses to ALA in different tissues.Similarly, the NO content in SNP-treated plants under salt stress was signi cantly higher than the non-salt control, but lower than that of NaCl alone in leaves (P < 0.05).This means that SNP inhibited NO increase in leaves but promoted it in roots under salt stress, a trend similar with ALA.On the contrary, addition with Na 2 WO 4 or cPTIO completely inhibited NO increase in strawberry (including leaves and roots), indicating that two inhibitors completely inhibited endogenous NO increase in strawberry plants under salt stress, induced by salt stress or ALA treatment.
When expressions of NR, nitrate reductase gene which is responsible for NO generation, were analyzed by qRT-PCR (Fig. 2B), the results showed that salt stress induced it up-expression by 96%, while ALA treatment induced more 4.25-fold compared with NaCl treatment.SNP had no effect on NR expression, while Na 2 WO 4 completely inhibited the gene expression induced by ALA.Thus, the increase of NO levels in roots induced by ALA might be dependent on the up-regulation of NR expression.
To con rm the promotive effect of ALA and NaCl on the NO content in roots of strawberry, the root tips were dyed by the speci c uorescent dyes DAF-FM DA and visualized using LSCM.No obvious NO uorescence signals can be seen in roots of the control, but it increased greatly when plants were subject to NaCl (Fig. 3).When co-treated with ALA, the NO uorescence further increased in the stele compared with NaCl alone.SNP treatment induced the release of NO, lling up the entire root section.However, when Na 2 WO 4 or cPTIO was added with ALA, the NO uorescence signal was negligible again compared with NaCl + ALA treatment.This suggests that two inhibitors eliminated root NO induced by ALA in saltstressed strawberry.

Effect of ALA and SNP on antioxidant enzyme activity of strawberry under salt stress
The SOD activity in leaves was much higher than that in roots, while the POD activity in roots was much higher than that in leaves, and the CAT activity in leaves was comparable with that in roots (Fig. 5).After NaCl stress, all three antioxidant enzymes activity increased signi cantly in both leaves and roots, and ALA or SNP treatment further promoted the increases.These suggest that ALA and NO possess similar ability to promote the activities of the antioxidant enzymes in strawberry under salt stress.When Na 2 WO 4 or cPTIO was co-treated with ALA, the increases of enzyme activities induced by ALA were all eliminated.This means that the promotion of ALA is dependent on NO presence.If NO generation was inhibited or scavenged, ALA did not induce increases of the antioxidant enzyme activities.It is worth noting that the effects of ALA on antioxidant enzyme activities in leaves and roots of strawberry are consistent, without tissue speci city.

Effect of ALA and SNP on the ion content in different tissues of strawberry under salt stress
The Na + content in strawberry was rather low under non-saline condition, which increased more than 17fold and 6-fold, respectively in leaves and roots after 100 mM NaCl treatment for 9 days (Fig. 6A).When ALA was co-treated, the leaf Na + content was decreased to 40% of NaCl alone, but 11% more accumulated in the roots (P < 0.05).It suggests that ALA has ability to induce more Na + interception in roots with less transport upward to leaves.When Na 2 WO 4 and cPTIO was co-treated with ALA, the leaf Na + content was 58%-64% higher than that without inhibitors, but in roots, the Na + interception induced by ALA was eliminated.These mean that ALA-induced Na + retention in roots is dependent on NO presence.When NO generation was blocked or eliminated, the root Na + retention was greatly impaired.
Nevertheless, effects of exogenous SNP treatment on Na + distribution was some different from ALA.The Na + content in leaves and roots of SNP-treated plants was only 46% and 66% of NaCl treatment alone, respectively.Thus, SNP induced almost half decrease of total Na + content in strawberry under salt stress, instead of Na + retention in roots.The effect of SNP was not completely consistent with ALA.Similar responses were found when the Cl -content was measured.It increased to 2.7-fold and 2.2-fold of the control, respectively in leaves and roots under salt stress.If ALA was co-treated with NaCl, the Cl - content in two tissues was 65% and 154% of NaCl treatment alone, respectively.Here, we found ALA treatment induced Cl -retention in roots with much less transport upward (Fig. 6B), quite similar with Na + .
When co-treated with Na 2 WO 4 and cPTIO, the ALA-induced Cl -retention in roots was eliminated.Thus, the effect of ALA on Cl -retention may be also dependent on NO presence.Interestingly, the effect of SNP treatment was not the same with ALA.The leaf Cl -content of SNP treatment was 69% of NaCl alone, similar with ALA treatment, whose content of Cl -in roots was also 70% of NaCl alone.Thus, the total Cl - content in SNP-treated plants was about 70% of NaCl alone.No Cl -interception in roots was found in SNP-treated plants, although the salt tolerance was signi cantly improved.Declination of salt uptake may be an important mechanism of salt tolerance induced by SNP.

Effects of ALA and SNP on gene expressions related with ion transporters
To understand molecular mechanisms underlying the role of ALA and NO in salt tolerance, we analyzed the expressions of genes related to Na + and Cl -transport.Four SOS expressions in strawberry roots tended to increase after NaCl treatment for 48 h, however, only SOS1 and SOS2 were signi cant at P = 0.05 (Fig. 7).ALA treatment greatly induced all four SOS gene expressions under salt stress, and SNP also up-regulated most of SOSs but not SOS4 expression.When Na 2 WO 4 or cPTIO was co-treated with ALA, the up-regulation of gene expressions (including SOS1, SOS2 and SOS4) by ALA was almost completely eliminated, suggesting these three gene up-expressions by ALA were dependent on NO presence.However, the up-expression of SOS3 by ALA might not be dependent on NO, because the gene expression in Na 2 WO 4 treatment was still 3.3 times as high as NaCl alone, and cPTIO did not inhibit the effect of ALA on SOS3 expression.Thus, there should be other routes besides NO signal when ALA induced SOS3 expression.The fact that ALA rather than SNP induced SOS4 up-expression and two inhibitors of NO eliminated ALA's effect suggests that NO may be one of essential inductive factors for SOS4 expression.
The responses of NHX1and HKT1 expressions to different treatments were quite consistent with each other (Fig. 7B).It seems that salt stress tended to up-regulate these gene expressions, however, the effect was not signi cant at 48 h after salt stress.ALA and SNP dramatically induced the gene expressions, while Na 2 WO 4 or cPTIO completely eliminated the promotion of ALA.These results suggest that ALAinduced gene expressions in strawberry roots was dependent on NO presence.
CLCs is a family of genes coding chloride channel proteins, mediating Cl -uptake, transport and compartmentation.In strawberry genome, we found four genes highly homologous with Arabidopsis and analyzed their expressions by qRT-PCR.Salt stress up-regulated all CLC expressions in strawberry roots (P < 0.05) and ALA further up-regulated the increases under salt stress (Fig. 7C).SNP also up-regulated the expressions of CLC-d, CLC-f and CLC-g but not CLC-a.Co-treatment with Na 2 WO 4 or cPTIO eliminated the promotion of ALA on CLC-a, CLC-d and CLC-f but not CLC-g.It seems that ALA treatment is su cient to up-regulate CLC-a expression, where NO is essential but not enough.Therefore, only SNP addition was not enough to induce CLC-a up-expression, while Na 2 WO 4 or cPTIO completely eliminated the effect of ALA.Additionally, ALA was su cient to up-regulate CLC-d and CLC-f expression, where NO was both su cient and essential.The responses of CLC-g expression were different from the formers.Its expression in ALA + Na 2 WO 4 treatment was much higher than ALA itself, implying that inhibition of NO generation did not block the gene expression induced by ALA.Similarly, the gene expression in ALA + cPTIO treatment was comparable with ALA only.Thus, the up-regulation of CLC-g expression by ALA cannot be eliminated by NO inhibitors, which suggests that both ALA and NO induced the gene expression, but ALA-induced regulation did not depend on NO presence.ALA-induced up-expression of CLC-g may be mediated through the other routes.

Discussion
The bene cial 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 H 2 O 2 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 signi cantly 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, ALAinduced 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].In present work, we once again observed that ALA treatment decreased the leaf Na + content but signi cantly increased the retention in the roots of strawberry (Fig. 6A).It is quite agreed with our previous ndings [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 signi cantly 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 H 2 O 2 on up-expressions of Na + transporter genes.H 2 O 2 is known as a reaction oxygen species (ROS), as well as an important cellular signal.However, in most studies about ALA, H 2 O 2 was considered as ROS rather than a cellular signal [23].Never attention had been payed to different responses of H 2 O 2 between shoots and roots [7] until Wu et al., who found that salt stress induced H 2 O 2 increase in both leaves and roots, and ALA induced more H 2 O 2 increase in roots but depressed in leaves [25].The tissue-speci c response is con rmed 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 O 2 • production rates in both leaves and roots strawberry under salt stress, however, there was no H 2 O 2 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 H 2 O 2 increase in roots of strawberry, while Na 2 WO 4 or cPTIO inhibit the H 2 O 2 increase induced by ALA (Fig. 4).This means that ALA-induced H 2 O 2 increase in roots of strawberry is dependent on NO presence.In another word, NO may be a signal located at the upstream of H 2 O 2 , involved in ALA-induced plant salt tolerance.However, in cucumber roots, ALA did not induce H 2 O 2 increase [54].The reason for this difference needs further clari cation.
It is the rst 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, Na 2 WO 4 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 signi cantly induced NR up-expression in roots of strawberry, while Na 2 WO 4 completely eliminated the expression induced by ALA (Fig. 2B).Thus, the inhibition of NR expression by Na 2 WO 4 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-speci c, similar with H 2 O 2 (Fig. 4).
We used DAF-FM DA, a highly speci c NO uorescent 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-H 2 O 2 signaling route in ALA regulating salt tolerance of strawberry.We have found that hematin, a CO donor can induce both increases of NO and H 2 O 2 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.Signi cant 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) 6 3-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 K 4 Fe(CN) 6 did not exhibit signi cant 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 Na 2 WO 4 or cPTIO (Fig. 5).These mean that ALA-induced antioxidant enzyme activity is dependent on NO presence.However, no tissue-speci c responses of antioxidant enzyme activity to ALA are found here.Thus, the response of H 2 O 2 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.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) signi cantly in strawberry roots, while Na 2 WO 4 or cPTIO eliminated the effect induced by ALA.Thus, we propose that NO is another necessary component in the signal cascade of ALAinduced salt tolerance of strawberry, at the up-stream of H 2 O 2 , 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 identi ed.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 nd out four genes of FaSOS family, where FaSOS1 and FaSOS2 can be coincidently upregulated by ALA and SNP under salt stress, but the effect is eliminated by Na 2 WO 4 or cPTIO (Fig. 7A).
These suggest that ALA as well as its induced NO is su cient 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 su cient 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 su cient.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 H 2 O 2 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 NO 3 -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 identi ed.For example, AtCLC-a as well as the highly homologues AtCLC-b, is responsible for NO 3 -/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 bene cial 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 signi cantly 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 Na 2 WO 4 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 speci c.Anyway, it is interesting to notice that all the gene upexpressions induced by ALA and NO seem to be bene cial 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.

Conclusion
Our results here have veri ed the previous nding [25] that ALA induces Na + preferable retention in roots of strawberry to avoid excess accumulation in leaves.Also, Cl -is preferably retained in roots with less transport to the aboveground after ALA treatment (Fig. 6).Thus, ALA-induced retention of Na + and Cl -in roots may be one of the most important mechanisms for plants to cope with toxic ions.In the signaling cascade of ALA regulation, we nd that ALA can induce NO and then H 2 O 2 accumulation in roots of strawberry, where NO functions at the up-stream of H 2 O 2 signaling, involved in up-regulating gene expressions for ion homeostasis under salt stress.We modi ed the hypothesis (Fig. 8) from the previous [25].We believe that it is one of the most attractive hypotheses about ALA regulating salt tolerance up to now.More detailed interactions at different levels need to be elucidated in the future.
Treatments were arranged in a completely randomized design with three replications.Five plants consisted of a block.Part of plants were harvested on the 2 nd day for gene expression analysis, part harvested on the 6 th day for physiological analysis, and the remains harvested on the 9 th day for growth and salty ion analysis.

Plant growth
Plant growth was measured in terms of dry weight of strawberry.Three plants were collected in a treatment, washed clear and divided into roots and shoots, oven dried at 105℃ for 10 min rstly, and then 70℃ for 72 h and weighed with a digital balance.

Measurement of NO levels
Endogenous NO content of strawberry after 6-days treatment was determined using the Greiss method with slight modi cations [77].Leaves and roots (0.5 g) were ground in a mortar in 5 mL of 40 mM cool HEPES buffer (pH 7.2) and little quartz sand.The homogenates were centrifuged at 8000 × g for 10 min at 4℃.One milliliter supernatant was mixed with 1 mL Greiss reagent, incubated at room temperature for 30 min.Absorbance was determined at 540 nm.NO content was calculated according to a standard curve of NaNO 2 .

Fluorescent imaging of NO in roots
NO in roots was visualized using the highly speci c NO uorescent probe 3-amino, 4-aminomethyl-2', 7'di uoro uorescein diacetate (DAF-FM DA), according to the method described by Corpas et al. [78].The slices of strawberry roots were incubated with 20 µM DAF-FM DA at 25℃ under darkness for 2 h, then washed clear using phosphate buffer solution (pH 7.4) to discard excess uorophore.DAF-FM DA uorescence was visualized using a Zessi Laser Scanning Confocal Microscope (LSCM) with 480 nm excitation and 535 nm emission lters.Ten individual roots of each treatment were observed, and uorescence intensities from about 50 cells were averaged for each treatment.
Determination of H 2 O 2 content H 2 O 2 content of strawberry after 6-days treatment was determined according to the method of Liu et al. [79] with slight modi cations.Leaves and roots (0.2 g) were ground in a mortar containing 5 mL of cool acetone and a little quartz sand.After 8000 × g centrifugation for 10 min, 1 ml of the supernatant was taken to mix with 3 ml mixture of CCl 4 ∶CHCl 3 (3∶1, V/V) and 5 ml distilled water to extract hydrophobic pigment.After 4000 × g centrifugation for 1 min, the water phase was taken for next reaction.In a reaction mixture, 1 mL of the supernatant, 1 mL of 5% titanium sulfate and 2 mL of strong ammonia were added in turn.After precipitate formed, the mixture was centrifuged at 8000 × g for 10 min to discard the supernatant.Then, 5 mL of sulfuric acid was added to dissolve the precipitation.Absorbance at 450 nm was determined with a spectrophotometer.

Assay of antioxidant enzymes
Fresh tissues (0.2 g) were ground in a mortar and pestle in 8 mL of 50 mM cool phosphate buffer (pH 7.8), containing 1% (w/v) polyvinyl pyrrolidone (PVP).The homogenates were centrifuged at 12000 × g for 15 min at 4℃.The supernatants were used for assays of enzyme activity Determination of Na + and Cl - About 0.2 g of dried powders of leaves and roots were put in a digestion tube containing 5 mL of HNO 3 for digestion in a micro-wave digestion system.The solution was transferred to a 50 mL volumetric ask and dilute with distilled water.The Na + concentrations were determined by an inductively coupled plasma optical emission spectrometer (ICP-OES 2100).Determination of Cl -content was referred to Lei et al. [83].About 0.2 g tissue powder was added to 20 mL of distilled water, shaken in the boiling water bath for 1 h and cooled.The solution was diluted to 50 mL with deionized water.Appropriate amount of Cl -test solution was taken in a 25 mL volumetric ask, mixed with 2 ml of HNO 3 solution (concentrated HNO 3 : deionized water = 3:1), 2 mL of acetone, 1 mL of 5 g L -1 AgNO 3 .The reaction mixture was nally dilute to 25 mL with deionized water.The absorbance was measured at 335 nm after placing it in the dark for 10 min.

RNA extraction and qRT-PCR
The roots of strawberry 48 h after treatment were used for gene expression analysis.Total RNA was extracted with CTAB method according to TransScriptÒ One-Stpe gDNA Removal instructions.RNA was reversely transcribed to produce cDNA using First-Strand cDNA synthesis kit (Transgen Biotech), and the resulting cDNA mixture was used as templates for subsequent PCRs.Real-time quantitative PCR was performed according to the manufacturer's instructions using the SYBR® Premix Ex TaqTM (Tli RNaseH Plus, TaKaRa, RR420A).Relative gene expression of mRNA was calculated using the 2 −ΔΔCT method [84].All qRT-PCR primers were listed in Table 1 with Actin as the internal control.Three biological replicates were prepared for each sample.

Statistical analysis
Each experiment repeated at least three times.Values in gures were expressed as means ± se.The statistical signi cance of the data was analyzed using a univariate analysis of variance (P < 0.05) (one- Thus, the ndings, 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]. Experiments were conducted in Nanjing Agricultural University.Strawberry (Fragaria × ananassa Duch.cv.'Benihoppe') plants with uniform size were transplanted to a disposable plastic cup lled with mixture medium (peat: vermiculite: perlite = 4: 2: 1, v: v: v, about 2/3 attached to the top of cup), with one plant in each cup.Plants were watered with 1/2 Hoagland nutrient solution every three days.When the 6 th leaf fully expanded, the plants were divided into six groups for following treatments, with15 plants in one group.(1) Control, each plant was poured with 200 mL 1/2 Hoagland solution.(2) NaCl, poured with 200 mL 1/2 Hoagland solution containing 100 mmol L -1 NaCl.(3) NaCl + ALA, poured with 200 mL 1/2 Hoagland solution containing 100 mmol L -1 NaCl and 10 mg L -1 ALA.(4) NaCl + SNP, poured with 200 mL 1/2 Hoagland solution containing 100 mmol L -1 NaCl and 10 µM sodium nitrosylpentacy (SNP, a donor of NO).(5) NaCl + ALA + Na 2 WO 4 , poured with 200 mL 1/2 Hoagland solution containing 100 mmol L -1 NaCl, 10 mg L -1 ALA and 5 µM Na 2 WO 4 , an inhibitor of nitrate reductase, which is responsible for NO generation, (6) NaCl + ALA + cPTIO, poured with 200 mL 1/2 Hoagland solution containing 100 mmol L -1 [80].Superoxide dismutase activity was determined according to Zhang et al. [81].Peroxidase and catalase activities were determined according to Chance & Maehly [82].

Figure 1 Comparison
Figure 1

Figure 3 The
Figure 3

Figure 4 Effect
Figure 4

Figure 7 Expressions
Figure 7 Effect of ALA and NO on the H 2 O 2 content in different tissues of strawberry under salt stress Strawberry leaves contained more H 2 O 2 than roots (Fig.4).Salt stress induced H 2 O 2 increase signi cantly in both of leaves and roots.The treatment with ALA or SNP completely eliminated the H 2 O 2 increase induced by salt stress in leaves but further promoted it in roots.This means that ALA and NO share a similar effect on H 2 O 2 levels, although the H 2 O 2 responses to ALA or SNP treatment under salinity are different between leaves and roots.Co-treatment of ALA with Na 2 WO 4 or cPTIO greatly depressed the endogenous H 2 O 2 content in the strawberry stressed by NaCl, which in the leaves was even signi cantly lower than NaCl treatment alone.Therefore, it can be deduced that H 2 O 2 accumulation induced by NaCl either with or without ALA may be dependent on the presence of NO, which probably acts on the upstream of H 2 O 2 signal route and participates in the induction of salt tolerance by ALA.
Correlation analysis shows that the root H 2 O 2 content was signi cantly positive correlated with the antioxidant enzyme activities in roots, where r SOD = 0.898 * , r POD = 0.944 ** , r CAT = 0.936 ** , respectively.However, the leaf H 2 O 2 content does not correlate with three enzyme activities (P > 0.05).It seems that the relations between H 2 O 2 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 H 2 O 2 was necessary for

Table 1
Primers of RT-PCR for strawberry genes related with salt stress