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Comparative effects of salt and alkali stresses on organic acid accumulation and ionic balance of seabuckthorn (Hippophae rhamnoides L.)
Industrial Crops and Products 30 (2009) 351–358
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Comparative effects of salt and alkali stresses on organic acid accumulation and ionic balance of seabuckthorn (Hippophae rhamnoides L.) Wanchao Chen, Pengjuan Cui, Huaying Sun, Weiqing Guo, Chunwu Yang, Hao Jin, Bin Fang, Decheng Shi ∗ Key Laboratory of Vegetation Ecology of MOE, Northeast Normal University, Changchun 130024, Jilin Province, China
a r t i c l e
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Article history: Received 2 May 2009 Received in revised form 25 June 2009 Accepted 30 June 2009
Keywords: Alkali stress Hippophae rhamnoides Organic acid Ion balance Salt stress
a b s t r a c t Seabuckthorn (Hippophae rhamnoides L.) is uniquely capable of growing well under various extreme environmental conditions, such as water deficit, salt stress, low temperature, and high altitude. It is of economic value and its berries are used in cosmetics and pharmaceutical products. In this study, we compared the effects of salt stresses (9:1 molar ratio of NaCl to Na2 SO4 , pH 6.48–6.65) and alkali stresses (9:1 molar ratio of NaHCO3 to Na2 CO3 , pH 8.70–8.88) on the levels of inorganic ions and organic acids in H. rhamnoides L. to elucidate the physiological mechanism by which it tolerates salt or alkali stress (high pH). The results showed that, in leaves and stems under alkali stress, the Na+ content increased to a much greater extent than under salt stress. Neither salt nor alkali stress decreased the K+ content in leaves and stems; however, in roots, the K+ content decreased sharply with increasing alkali stress, whereas it remained relatively unchanged with increasing salt stress. This revealed a specific mechanism of absorption or transport for Na+ and K+ that was affected strongly by alkali stress. The results indicated that accumulation of organic acid (OA) was a central adaptive mechanism by which H. rhamnoides maintained intracellular ionic balance under alkali stress. OA may play different roles in different organs during adaptation to alkali stress, and its percentage contribution to total negative charge was higher in leaf than in stem. H. rhamnoides accumulated mainly malate, oxalate, and citrate in leaves and stems; however, in roots, less malate and citrate was accumulated, and acetate accumulation was enhanced significantly, which indicated that roots and shoots use different mechanisms to modulate OA metabolism. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The existence of alkali stress has been demonstrated clearly by a number of reports, which have shown it to be more severe than salt stress (Campbell and Nishio, 2000; Hartung et al., 2002; Shi and Sheng, 2005; Shi and Wang, 2005; Yang et al., 2007, 2008a,b,c; Gao et al., 2008). In previous reports, we suggested that salt stress be defined as the stress of neutral salts; and alkali stress as the stress of alkaline salts (Shi and Yin, 1993; Shi and Wang, 2005; Shi and Sheng, 2005). In some areas alkalization of the soil due to NaHCO3 and Na2 CO3 may be a more severe problem than soil salinization caused by neutral salts such as NaCl and Na2 SO4 . For example, in northeast China, more than 70% of the land area is alkaline grassland (Kawanabe and Zhu, 1991), and only a few alkalitolerant halophytes can survive (Zheng and Li, 1999). However, to date, research into salt stress has emphasized NaCl as the main contributing factor (Munns and Tester, 2008), and little attention has been paid to alkali stress (Shi et al., 2002; Shi and Wang, 2005; Shi
∗ Corresponding author. Tel.: +86 431 85269590; fax: +86 431 85684009. E-mail address: [email protected] (D. Shi). 0926-6690/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2009.06.007
and Sheng, 2005; Wang et al., 2007; Yang et al., 2008a,b,c; Gao et al., 2008). Stress due to soil salinity generally involves osmotic stress and ion-induced injury (Munns, 2002). Comparison of alkali stress with salt stress reveals an added effect of alkali stress due to high pH. The high pH environment that surrounds the roots can cause metal ions and phosphorus to precipitate (Shi and Zhao, 1997), can greatly affect absorption of inorganic anions such as Na+ , K+ , Cl− , NO3 − and H2 PO4 − , and can disrupt the ionic balance and pH homeostasis in tissues (Yang et al., 2007, 2008b). Thus, plants in alkaline soil must cope with both physiological drought and ion toxicity, and also maintain intracellular ionic balance. A striking feature of plant tissues is that the total content of organic acids (OAs) is higher than in other organisms. OAs have a potential role as metabolically-active solutes in osmotic adjustment, balance of cation excess, and pH homeostasis (LópezBucio et al., 2000). In recent years, reports have shown that some alkali-tolerant halophytes accumulate high concentrations of OAs under alkali stress (Yang et al., 2007, 2008b), but neither alkalisensitive maize (Qu and Zhao, 2004) nor the halophyte Suaeda sals, which has weak alkali tolerance (Qu and Zhao, 2003) accumulate OAs. These reports demonstrated unquestionably that OAs are
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important in the mechanism of alkali tolerance, especially in maintaining intracellular ionic balance. Seabuckthorn (Hippophae rhamnoides L.) is a thorny nitrogenfixing deciduous shrub that is distributed naturally in Asia and Europe (Raffo et al., 2004). Since ancient times, seabuckthorn berries have been used as a medicinal ingredient in Asian countries. It has been domesticated in some parts of the world. Seabuckthorn is a unique and valuable plant currently being investigated for its medicinal values all over the world (Purushothaman et al., 2008). Seabuckthorn berries are an excellent source of phytochemicals such as ascorbic acid, tocopherols, unsaturated fatty acids, phenols, and carotenoids. The berries have been used for the treatment of radiation damage, burns, oral inflammation, and gastric ulcers (Zadernowskia et al., 2005). Earlier studies have reported the protective effects of the seed oil against liver injury, atopic dermatitis and atherosclerosis (Purushothaman et al., 2008). Other positive health effects that have been claimed include reduction of plasma cholesterol levels, inhibition of platelet aggregation, and regulation of immune function (Zadernowskia et al., 2005). In addition, seabuckthorn is uniquely capable of growing well under extreme environmental conditions such as water deficit, low temperature, salt stress, and high altitude (Xu et al., 2009). It can grow well in soil with a pH >9 and salt content >0.5%. In China, seabuckthorn has been used widely in the restoration of salt-alkaline grassland and forests. In this study, we compared the effects of salt stresses (9:1 molar ratio of NaCl to Na2 SO4 , pH 6.48–6.65) and alkali stresses (9:1 molar ratio of NaHCO3 to Na2 CO3 , pH 8.70–8.88) on the inorganic ion content and accumulation of organic acids in seabuckthorn to elucidate the mechanism by which plants adapt to alkali stress (high pH). 2. Materials and methods 2.1. Plant materials Seeds of seabuckthorn (H. rhamnoides L.) were collected from alkaline grassland in western Jilin Province, China. The seeds were sown in 17-cm diameter plastic pots that contained 2.5 kg of washed sand. The pots were watered sufficiently with Hoagland nutrient solution every 2 d. Distilled water was used to compensate for evaporation at other times. All pots were placed outdoors and protected from rain. Temperatures during the experimental period were 24–28 ◦ C during the day and 20–23 ◦ C at night. 2.2. Design of simulated salt and alkaline conditions Two neutral salts were mixed in a 9:1 molar ratio (NaCl:Na2 SO4 ), and applied to the salt stress group. For the alkali stress group, two alkaline salts were mixed in a 9:1 molar ratio (NaHCO3 :Na2 CO3 ). Within each group, three total salt concentrations were applied: 30, 60, and 90 mM. The salt stress groups were labeled S1–S3 and the alkali stress groups A1–A3. Therefore, in the 90 mM solution for salt stress, a mixture of 81 mM NaCl and 9 mM Na2 SO4 resulted in ion concentrations of 99 mM Na+ , 81 mM Cl− , and 9 mM SO4 2− . In the 90 mM solution for alkali stress, a mixture of 81 mM NaHCO3 and 9 mM Na2 CO3 resulted in ion concentrations of 99 mM Na+ , 81 mM HCO3 − , and 9 mM CO3 2− . In the salt stress and alkali stress groups, the pH was 6.48–6.65 and 8.70–8.88, respectively. 2.3. Stress treatments The stress treatments were applied when the seedlings were eight weeks old. Twenty-eight pots of uniformly growing seedlings were divided randomly into seven sets, four pots per set. Each pot was considered a single replicate. One set was used as an untreated
control, and the remaining six sets were treated with the various stress treatments. The pots were watered thoroughly daily at 17:00–18:00 h with nutrient solution that contained the appropriate stress salts. Control plants were maintained by watering with nutrient solution. The entire duration of treatment was 10 d. 2.4. Measurement of physiological indices Plants were harvested in the morning after the final treatment, and were first washed with tap water followed by distilled water. Roots, stems, and leaves were separated and freeze-dried. Dry samples of plant material (100 mg) were treated with 20 mL of deionized water at 100 ◦ C for 1 h, and the resulting extract was used to determine the contents of free inorganic ions and organic acids. The content of NO3 − , Cl− , SO4 2− , H2 PO4 − and oxalic acid was determined by ion chromatography using a DX-300 ion chromatographic system with an AS4A-SC ion-exchange column and a CDM-II electrical conductivity detector (mobile phase: Na2 CO3 /NaHCO3 = 1.7/1.8 mM; DIONEX, Sunnyvale, USA). The levels of other OAs were also determined by ion chromatography using the DX-300 ion chromatographic system with an ICE-AS6 ion-exclusion column, CDM-II electrical conductivity detector, and an AMMS-ICE II MicroMembrane suppressor (mobile phase: 0.4 mM heptafluorobutyric acid; DIONEX, Sunnyvale, USA). An atomic absorption spectrophotometer (TAS-990, Purkinje General, Beijing, China) was used to determine the levels of Na+ , K+ , Ca2+ , and Mg2+ . 2.5. Statistical data analysis Measurements were obtained from four replicates. Data were analyzed by one-way analysis of variance (ANOVA) using the statistical software SPSS 13.0. The mean values for the different treatments were compared by the post hoc least significant difference (LSD) test. Values of P < 0.05 were considered to indicate significance. 3. Results 3.1. Cations With increasing stress, the Na+ content of both leaves and stems increased (Fig. 1A and B, P < 0.05), and the increments under alkali stress were greater than those under salt stress. Neither increasing salt stress nor increasing alkali stress reduced the K+ content in H. rhamnoides leaves and stems; however, in roots, the K+ content decreased sharply with increasing alkali stress (Fig. 1D and E, P < 0.05). Alkali stress enhanced Ca2+ accumulation in leaves and stems, whereas salt stress reduced Ca2+ accumulation by a small amount (Fig. 1G–I, P < 0.05). The response of Mg2+ to both stresses was similar (Fig. 1J–L). With increasing salinity, the Mg2+ content in leaves and stems remained unchanged, whereas it decreased a little in roots. 3.2. Anions Under salt stress, the Cl− content increased with increasing salinity (P < 0.05; Fig. 2A–C). With increasing alkali stress, the Cl− content in leaves and stems remained unchanged, whereas it decreased a little in roots (P < 0.05; Fig. 2A–C). Salt stress affected the NO3 − content only slightly (Fig. 2). However, alkali stress, especially strong alkali stress, affected NO3 − accumulation clearly: the NO3 − content in leaves and stems increased but it decreased in roots (Fig. 2D–F). The H2 PO4 − content under alkali stress was lower than that under salt stress (Fig. 2G–I). Under salt stress, with increasing salinity, the SO4 2− content in the leaf and stem both remained relatively unchanged, whereas it increased slightly in the
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Fig. 1. Effects of salt and alkali stresses on contents of Na+ , K+ , Ca2+ , and Mg2+ in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 : Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05).
Table 1 Percentage contribution of seven organic acids to total moles of organic acid in H. rhamnoides L. leaves under salt and alkali stresses. Salinity (mM)
Malate (%)
Citrate (%)
Succinate (%)
Acetate (%)
Oxalate (%)
Formate (%)
Lactate (%)
Salt stress
Control 30 60 90
29.50 24.15 19.33 19.30
21.13 18.61 17.70 18.06
2.44 2.18 2.20 1.82
4.57 5.12 6.11 5.68
34.82 41.74 45.45 46.48
4.08 4.66 5.50 5.71
3.46 3.54 3.71 2.96
Alkali stress
30 60 90
35.16 33.32 37.62
18.39 17.06 19.18
2.63 2.51 2.48
4.52 4.21 2.48
33.43 33.49 30.10
3.48 3.42 2.89
2.38 5.99 5.25
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Table 2 Percentage contribution of seven organic acids to total moles of organic acid in H. rhamnoides L. stems under salt and alkali stresses. Salinity (mM)
Malate (%)
Citrate (%)
Succinate (%)
Acetate (%)
Oxalate (%)
Formate (%)
Lactate (%)
Salt stress
Control 30 60 90
26.73 19.76 19.31 16.99
15.94 16.19 15.63 15.68
0.85 0.87 0.76 0.73
8.77 11.76 11.11 11.17
41.59 44.52 47.22 48.99
4.19 4.53 4.09 3.95
1.95 2.37 1.89 2.50
Alkali stress
30 60 90
28.71 33.10 37.39
15.12 15.42 15.85
1.25 1.16 1.42
8.12 7.59 5.92
41.04 38.57 35.62
3.88 2.20 2.12
1.89 1.97 1.68
Table 3 Percentage contribution of seven organic acids to total moles of organic acid in H. rhamnoides L. roots under salt and alkali stresses. Salinity (mM)
Salt stress
Control 30 60 90
Alkali stress
30 60 90
Malate (%)
Citrate (%)
Succinate (%)
Acetate (%)
Oxalate (%)
Formate (%)
Lactate (%)
5.94 3.45 3.51 5.46
10.92 10.52 12.83 13.41
0 0 0 0
24.39 23.06 21.41 17.48
48.07 50.14 51.72 52.12
7.38 7.90 5.07 6.40
3.29 4.93 5.46 5.12
8.06 11.64 6.54
20.33 18.15 15.23
0 0 0
32.99 31.87 53.47
31.57 25.47 15.26
4.92 9.49 3.02
2.13 3.38 6.48
root (Fig. 2J–L). However, alkali stress increased the SO4 2− content in the leaf and decreased the SO4 2− content in the root, but did not affect SO4 2− accumulation in the stem (Fig. 2J–L). 3.3. Organic acids Malate, citrate, succinate, acetate, oxalate, formate, and lactate were detected in the leaves and stems of H. rhamnoides (Tables 1 and 2). In addition, malate, citrate, acetate, oxalate, formate, and lactate were detected in the root (Table 3). Salt stress affected the accumulation of OAs only slightly (Figs. 3 and 4), and even reduced OA accumulation in leaves (Fig. 4J). However, alkali stress had a large effect on OA accumulation. Alkali stress, especially strong alkali stress, clearly increased the levels of OAs in leaves and stems, but reduced OA accumulation in the root (Figs. 3 and 4). The levels of OAs in leaves and stems were much higher than those in the root (Figs. 3 and 4). Oxalate, malate, and citrate were the dominant OAs in leaves and stems (Tables 1 and 2). However, in roots, less malate and citrate were accumulated, whereas acetate accumulation was enhanced significantly (Fig. 3 and Table 3). Remarkably, strong alkali stress caused acetate to be the dominant component; it comprised 53.47% of the total OAs in roots (Fig. 4D–F and Table 3). 4. Discussion 4.1. Inorganic ions It has been reported that salt stress and alkali stress are actually two distinct types of stress and that the injurious effects of alkali stress on plants are more severe than those of salt stress (Yang et al., 2007, 2008a,b,c, 2009). Alkali stress involves the same stress factors as salt stress but with the added involvement of high pH. Adverse effects on roots are the basis of high pH stress (Yang et al., 2008a,b,c, 2009). The results reported here suggested that alkali stress (high pH) had a significant effect on the distribution and accumulation of inorganic ions in H. rhamnoides. Na+ is the main toxic ion in salinized soil. Low Na+ and high K+ in the cytoplasm are essential for the maintenance of a number of enzymatic processes (Munns and Tester, 2008). Na+ enters plant cells through the high-affinity K+ transporter (HKT) and through non-selective cation channels (Zhu, 2003). The similarity in size of the hydrated ionic radii of Na+ and K+ makes them difficult to discriminate between, and this is the basis of Na+ toxicity (Blumwald,
2000). Under salt stress, Na+ competes with K+ for uptake into roots (Munns, 2002; Munns and Tester, 2008). However, our results showed that neither increasing salt stress nor increasing alkali stress decreased the K+ content in H. rhamnoides leaves and stems; although, in roots, the K+ content decreased sharply with increasing alkali stress (Fig. 1). This phenomenon demonstrated that no competition occurred between Na+ and K+ absorption, which suggested that H. rhamnoides might have a unique pathway of Na+ absorption. Under alkali stress, the decreased K+ content in the root might be attributable to an inhibitory effect of high pH on K+ absorption, which relies on the transmembrane proton gradient (Munns, 2002; Munns and Tester, 2008). In addition, in leaves and stems, the increase in Na+ under alkali stress was much higher than that under salt stress (Fig. 1). We propose that this was not a response to osmotic stress, but a specific response to high pH stress. Alkali stress might weaken the controls on absorption or transport of Na+ , which in turn would lead to the sharp increase in Na+ content in leaves and stems (Fig. 1A), and disrupt the ionic balance or pH homeostasis in tissue. The increased level of Na+ in shoots under alkali stress might also be related to decreased exclusion of Na+ . It is well known that many plant species have a Na+ exclusion mechanism that depends on a Na+ /H+ antiporter, such as salt overly sensitive 1 (SOS1), which exchanges cytoplasmic Na+ for external H+ (Zhu, 2003; Munns and Tester, 2008). The exchange activity relies on the transmembrane proton gradient, which is established by H+ -ATPase (Zhu, 2003). Under strong alkali stress, the lack of external protons might weaken the exchange activity of the Na+ /H+ antiporter in the root plasma membrane (Munns and Tester, 2008), and possibly reduce exclusion of Na+ into the rhizosphere and enhance in vivo accumulation of Na+ . Neither alkali stress nor salt stress affected Mg2+ accumulation in H. rhamnoides. The response of free Ca2+ to each stress was interesting. Salt stress did not affect accumulation of Ca2+ in leaves and stems, but alkali stress did cause the Ca2+ content to increase (Fig. 1). However, in roots, both salt and alkali stress decreased the Ca2+ content (Fig. 1). This might be a strain-specific response of H. rhamnoides to alkali stress (high pH), whose unknown cause and function should be investigated further. Given that nitrogen is one of the most essential elements in all biological materials, changes in its nutrition and metabolism are of particular importance. NO3 − plays an important role in the maintenance of intracellular ionic balance and osmotic adjustment when plants are under salt or alkali stress (Yang et al., 2007, 2008b, 2009).
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Fig. 2. Effects of salt and alkali stresses on contents of Cl− , SO4 2− , NO3 − and H2 PO4 − in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 :Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05). Table 4 Percentage of the contribution of various free anions to total negative charge in H. rhamnoides L. tissue under salt and alkali stresses. Salinity (mM)
Salt stress
Control 30 60 90
Alkali stress
30 60 90
Leaf
Stem
Cl− (%)
SO4 2− (%)
NO3 − (%)
H2 PO4 − (%)
OA (%)
Cl− (%)
SO4 2− (%)
NO3 − (%)
H2 PO4 − (%)
OA (%)
10.07 17.95 22.16 22.88
8.72 8.80 8.81 9.22
1.71 1.54 1.16 1.00
8.73 8.09 8.72 8.34
70.77 63.61 59.14 58.57
10.22 15.62 19.39 22.00
18.65 15.61 14.20 14.05
2.18 1.59 1.51 1.12
13.88 14.26 14.51 14.71
55.07 52.92 50.39 48.11
7.53 6.81 6.29
7.55 9.45 8.25
1.19 1.50 2.34
5.28 4.41 3.91
78.46 77.83 79.22
8.71 7.92 7.44
12.29 10.56 9.46
1.46 2.51 4.36
10.32 8.46 8.49
67.22 70.55 70.25
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Fig. 3. Effects of salt and alkali stresses on contents of malate, citrate, formate and succinate in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 :Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05).
The effects of salt or alkali stress on NO3 − content in H. rhamnoides were interesting. Salt stress did not affect the accumulation of NO3 − , whereas it was affected strongly by alkali stress (Fig. 2). Under alkali stress, as salinity increased, the NO3 − content in both leaves and stems increased, but it decreased in roots (Fig. 2). Although we did not measure other physiological parameters involved in nitrogen metabolism, the changes in NO3 − content clearly indicated that alkali stress can interfere with the uptake or metabolism of NO3 − . We assumed that NO3 − was deoxidized in the root because the NO3 − content in the H. rhamnoides root was much higher than that in the shoot. It has been proposed that NO3 − uptake is mediated by a 2H+ /1NO3 − symport mechanism, which relies on the transmembrane proton gradient (Crawford and Glass, 1998). The reduction
in NO3 − in the root under alkali stress might be related to the lack of external protons due to the high pH, and should be investigated further. 4.2. Ionic balance and accumulation of organic acids Ionic imbalance in plants is caused mainly by the influx of excess Na+ (Munns and Tester, 2008; Yang et al., 2007). Plants in saline conditions usually accumulate inorganic anions such as Cl− , NO3 − , H2 PO4 − , and SO4 2− or organic anions to neutralize the high concentrations of cations and maintain a stable intracellular pH (Yang et al., 2007, 2008b). As shown in Table 4, different anions differed in their contribution to the overall negative charge. The percentage contri-
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Fig. 4. Effects of salt and alkali stresses on contents of lactate, acetate, oxalate, total organic acid (OA) in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 :Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05).
bution of various anions to the total negative charge ranged from high to low as follows: OAs, Cl− , SO4 2− , H2 PO3 − , NO3 − . Although OA was the dominant factor in maintaining ionic equilibrium in stressed H. rhamnoides, its percentage contribution to total negative charge under alkali stress was higher than under salt stress, and in addition was higher in leaves than in stems (Table 4). This implied that OA may play different roles during different types of stress or in different tissues. Under alkali stress, especially strong alkali stress, the synthesis of OAs was enhanced in H. rhamnoides to neutralize the massive influx of Na+ into leaves and stems (Fig. 1). Na+ might participate in the signal transduction pathway by which OA synthesis is induced during the response to alkali stress, and this should be investigated in future. During the response to alkali stress, different OAs had different roles in maintaining the intracellular ionic balance. This showed
that accumulation of OAs was not simply a passive response to a negative charge deficit, but was a result of active metabolic regulation after alkali stress had been sensed. The metabolic regulation of OAs under alkali stress may involve one or more key enzymes. These key enzymes may be known enzymes that participate in basal metabolic pathways such as the tricarboxylic acid cycle, glyoxylate cycle, glycolysis or other pathways. Enzyme activity can be regulated at the level of synthesis (transcription, translation and modification of new polypeptides), or after synthesis by the action of activators and inhibitors. If the pattern of OA accumulation in response to alkali stress can be defined clearly, it should be possible to identify and clone the key genes of plant alkali tolerance easily. This requires further investigation. In summary, OA accumulation is a central adaptive mechanism by which H. rhamnoides maintain intracellular ionic balance under
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alkali stress. H. rhamnoides accumulated three main OAs (oxalate, malate, and citrate) in leaves and stems (Tables 1 and 2). Comparing these results with those of our previous studies, the mechanism by which OA metabolism was modulated in H. rhamnoides was significantly different from that in Chenopodiaceae halophytes such as Kochia sieversiana (Yang et al., 2007) and Suaeda glauca (Yang et al., 2008b). In both the latter two species, the accumulated oxalate represents approximately 90% of total OA, under both alkali and salt stress. These data indicate that the modulation of OA metabolism may play different roles in different plant species. However, changes in OA metabolism are still an important in vivo adaptive response of halophytes to alkali stress, and should constitute an important future direction for research into the physiology of plant alkali stress. As mentioned previously, H. rhamnoides accumulated mainly malate, oxalate, and citrate in leaves and stems (Figs. 3 and 4, Tables 1 and 2). However, in roots, less malate and citrate accumulated, and acetate accumulation was enhanced significantly (Figs. 3 and 4, Table 3), which indicated that roots and shoots might use different mechanisms to modulate OA metabolism. As an alkali-tolerant plant, H. rhamnoides may have evolved unique mechanisms to regulate OA metabolism. Under alkali stress, especially strong alkali stress, H. rhamnoides may enhance the rate of anaerobic respiration, which would increase synthesis of acetate. Thus, H. rhamnoides may have a strong capacity to tolerate hypoxic conditions, which could contribute to its high alkali tolerance. It is well known that alkali stress can destroy soil structure and greatly decrease soil permeability, which can impose hypoxic stress. As a result of long-term adaptation to hypoxic conditions in its habitat (alkaline soil), H. rhamnoides may have evolved specific mechanisms to tolerate hypoxic stress. These should be investigated further. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 30671491) and the National Key Basic Research Program of China (No. 2007CB106800). We thank International Science Editing (ISE) from Ireland for language editing. References Blumwald, E., 2000. Sodium transport and salt tolerance in plants. Curr. Opin. Cell Biol. 12, 431–434. Campbell, S.A., Nishio, J.N., 2000. Iron deficiency studies of sugar beet using an improved sodium bicarbonate-buffered hydroponics growth system. J. Plant Nutr. 23, 741–757. Crawford, N.M., Glass, A.D.M., 1998. Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci. 3, 389–395. Gao, C., Wang, Y., Liu, G., Yang, C., Jiang, J., Li, H., 2008. Expression profiling of salinity–alkali stress responses by large-scale expressed sequence tag analysis in Tamarix hispid. Plant Mol. Biol. 66, 245–258. Hartung, W., Leport, L., Ratcliffe, R.G., Sauter, A., Duda, R., Turner, N.C., 2002. Abscisic acid concentration, root pH and anatomy do not explain growth differences
of chickpea (Cicer arietinum L.) and lupin (Lupinus angustifolius L.) on acid and alkaline soils. Plant Soil 240, 191–199. Kawanabe, S., Zhu, T.C., 1991. Degeneration and conservation of Aneurolepidium chinense grassland in Northern China. J. Jpn. Grassland Sci. 37, 91– 99. López-Bucio, J., Nieto-Jacobo, M.F., Ramírez-Rodríguez, V., Herrera-Estrella, L., 2000. Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant Sci. 160, 1–13. Munns, R., 2002. Comparative physiology of salt and water stress. Plant Cell Environ. 25, 239–250. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651–681. Purushothaman, J., Suryakumar, G., Shukla, D., Malhotra, A.S., Kasiganesan, H., Kumar, R., Sawhney, R.C., Chami, A., 2008. Modulatory effects of seabuckthorn (Hippophae rhamnoides L.) in hypobaric hypoxia induced cerebral vascular injury. Brain Res. Bull. 77, 246–252. Qu, Y.G., Zhao, K.F., 2003. Comparison of the stress effects of NaCl and Na2 CO3 on Suaeda salsa. Chin. J. Plant Physiol. Mol. Biol. 29, 387–394. Qu, Y.G., Zhao, K.F., 2004. Comparative studies on growth and physiological reaction of Zea mays under NaCl and Na2 CO3 stress. Chin. Acta Agron. Sin. 30, 334– 341. Raffo, A., Paoletti, F., Antonelli, M., 2004. Changes in sugar, organic acid, flavonol and carotenoid composition during ripening of berries of three seabuckthorn (Hippophae rhamnoides L.) cultivars. Eur. Food Res. Technol. 219, 360–368. Shi, D.C., Zhao, K.F., 1997. Effects of NaCl and Na2 CO3 on growth of Puccinellia tenuiflora and on present state of mineral elements in nutrient solution. Acta Pratacu. Sin. 6, 51–61. Shi, D.C., Yin, L.J., 1993. Difference between salt (NaCl) and alkaline (Na2 CO3 ) stresses on Puccinellia tenuiflora (Griseb) Scribn et Merr. plants. Acta Bot. Sin. 3, 144– 149. Shi, D.C., Yin, S.J., Yang, G.H., Zhao, K.F., 2002. Citric acid accumulation in an alkalitolerant plant Puccinellia tenuiflora under alkaline stress. Acta Bot. Sin. 44, 537–540. Shi, D.C., Wang, D., 2005. Effects of various salt–alkali mixed stresses on Aneurolepidium chinense (Trin) Kitag. Plant Soil 271, 15–26. Shi, D.C., Sheng, Y., 2005. Effect of various salt–alkaline mixed stress conditions on sunflower seedlings and analysis of their stress factors. Environ. Exp. Bot. 54, 8–21. Wang, Y., Yang, C., Liu, G., Jiang, J., 2007. Development of a cDNA microarray to identify gene expression of Puccinellia tenuiflora under saline–alkali stress. Plant Physiol. Biochem. 45, 567–576. Xu, G., Li, C., Yao, Y., 2009. Proteomics analysis of drought stress-responsive proteins in Hippophae rhamnoides L. Plant. Mol. Biol. Rep. 27, 153–161. Yang, C., Chong, J., Kim, C., Li, C., Shi, D., Wang, D., 2007. Osmotic adjustment and ion balance traits of an alkali resistant halophyte Kochia sieversiana during adaptation to salt and alkali conditions. Plant Soil 294, 263–276. Yang, C., Jianaer, A., Li, C., Shi, C., Wang, D., 2008a. Comparison of the effects of salt-stress and alkali-stress on the photosynthetic production and energy storage of an alkali-resistant halophyte Chloris virgata. Photosynthetica 46, 273– 278. Yang, C., Shi, D., Wang, D., 2008b. Comparative effects of salt stress and alkali stress on growth, osmotic adjustment and ionic balance of an alkali resistant halophyte Suaeda glauca (Bge). Plant Growth Regul. 56, 179–190. Yang, C., Wang, P., Li, C., Shi, D., Wang, D., 2008c. Comparison of effects of salt and alkali stresses on the growth and photosynthesis of wheat. Photosynthetica 46, 107–114. Yang, C., Xu, H., Wang, L., Liu, J., Shi, D., Wang, D., 2009. Comparative effects of saltstress and alkali-stress on the growth, photosynthesis, solute accumulation, and ion balance of barley plants. Photosynthetica 47, 79–86. Zadernowskia, R., Naczk, M., Czaplickia, S., Rubinskiene, M., Szalkiewicz, M., 2005. Composition of phenolic acids in sea buckthorn (Hippophae rhamnoides L.) berries. J. Am. Oil Chem. Soc. 82, 175–179. Zheng, H.Y., Li, J.D., 1999. Form and dynamic trait of halophyte community. In: Zheng, H.Y. (Ed.), Saline Plants in Songnen Plain and Restoration of Alkaline–Saline Grass. Science Press, Beijing, pp. 137–138. Zhu, J.K., 2003. Regulation of ion homeostasis under salt stress. Curr. Opin. Cell Biol. 6, 441–445.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Comparative effects of salt and alkali stresses on organic acid accumulation and ionic balance of seabuckthorn (Hippophae rhamnoides L.) Wanchao Chen, Pengjuan Cui, Huaying Sun, Weiqing Guo, Chunwu Yang, Hao Jin, Bin Fang, Decheng Shi ∗ Key Laboratory of Vegetation Ecology of MOE, Northeast Normal University, Changchun 130024, Jilin Province, China
a r t i c l e
i n f o
Article history: Received 2 May 2009 Received in revised form 25 June 2009 Accepted 30 June 2009
Keywords: Alkali stress Hippophae rhamnoides Organic acid Ion balance Salt stress
a b s t r a c t Seabuckthorn (Hippophae rhamnoides L.) is uniquely capable of growing well under various extreme environmental conditions, such as water deficit, salt stress, low temperature, and high altitude. It is of economic value and its berries are used in cosmetics and pharmaceutical products. In this study, we compared the effects of salt stresses (9:1 molar ratio of NaCl to Na2 SO4 , pH 6.48–6.65) and alkali stresses (9:1 molar ratio of NaHCO3 to Na2 CO3 , pH 8.70–8.88) on the levels of inorganic ions and organic acids in H. rhamnoides L. to elucidate the physiological mechanism by which it tolerates salt or alkali stress (high pH). The results showed that, in leaves and stems under alkali stress, the Na+ content increased to a much greater extent than under salt stress. Neither salt nor alkali stress decreased the K+ content in leaves and stems; however, in roots, the K+ content decreased sharply with increasing alkali stress, whereas it remained relatively unchanged with increasing salt stress. This revealed a specific mechanism of absorption or transport for Na+ and K+ that was affected strongly by alkali stress. The results indicated that accumulation of organic acid (OA) was a central adaptive mechanism by which H. rhamnoides maintained intracellular ionic balance under alkali stress. OA may play different roles in different organs during adaptation to alkali stress, and its percentage contribution to total negative charge was higher in leaf than in stem. H. rhamnoides accumulated mainly malate, oxalate, and citrate in leaves and stems; however, in roots, less malate and citrate was accumulated, and acetate accumulation was enhanced significantly, which indicated that roots and shoots use different mechanisms to modulate OA metabolism. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The existence of alkali stress has been demonstrated clearly by a number of reports, which have shown it to be more severe than salt stress (Campbell and Nishio, 2000; Hartung et al., 2002; Shi and Sheng, 2005; Shi and Wang, 2005; Yang et al., 2007, 2008a,b,c; Gao et al., 2008). In previous reports, we suggested that salt stress be defined as the stress of neutral salts; and alkali stress as the stress of alkaline salts (Shi and Yin, 1993; Shi and Wang, 2005; Shi and Sheng, 2005). In some areas alkalization of the soil due to NaHCO3 and Na2 CO3 may be a more severe problem than soil salinization caused by neutral salts such as NaCl and Na2 SO4 . For example, in northeast China, more than 70% of the land area is alkaline grassland (Kawanabe and Zhu, 1991), and only a few alkalitolerant halophytes can survive (Zheng and Li, 1999). However, to date, research into salt stress has emphasized NaCl as the main contributing factor (Munns and Tester, 2008), and little attention has been paid to alkali stress (Shi et al., 2002; Shi and Wang, 2005; Shi
∗ Corresponding author. Tel.: +86 431 85269590; fax: +86 431 85684009. E-mail address: [email protected] (D. Shi). 0926-6690/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2009.06.007
and Sheng, 2005; Wang et al., 2007; Yang et al., 2008a,b,c; Gao et al., 2008). Stress due to soil salinity generally involves osmotic stress and ion-induced injury (Munns, 2002). Comparison of alkali stress with salt stress reveals an added effect of alkali stress due to high pH. The high pH environment that surrounds the roots can cause metal ions and phosphorus to precipitate (Shi and Zhao, 1997), can greatly affect absorption of inorganic anions such as Na+ , K+ , Cl− , NO3 − and H2 PO4 − , and can disrupt the ionic balance and pH homeostasis in tissues (Yang et al., 2007, 2008b). Thus, plants in alkaline soil must cope with both physiological drought and ion toxicity, and also maintain intracellular ionic balance. A striking feature of plant tissues is that the total content of organic acids (OAs) is higher than in other organisms. OAs have a potential role as metabolically-active solutes in osmotic adjustment, balance of cation excess, and pH homeostasis (LópezBucio et al., 2000). In recent years, reports have shown that some alkali-tolerant halophytes accumulate high concentrations of OAs under alkali stress (Yang et al., 2007, 2008b), but neither alkalisensitive maize (Qu and Zhao, 2004) nor the halophyte Suaeda sals, which has weak alkali tolerance (Qu and Zhao, 2003) accumulate OAs. These reports demonstrated unquestionably that OAs are
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important in the mechanism of alkali tolerance, especially in maintaining intracellular ionic balance. Seabuckthorn (Hippophae rhamnoides L.) is a thorny nitrogenfixing deciduous shrub that is distributed naturally in Asia and Europe (Raffo et al., 2004). Since ancient times, seabuckthorn berries have been used as a medicinal ingredient in Asian countries. It has been domesticated in some parts of the world. Seabuckthorn is a unique and valuable plant currently being investigated for its medicinal values all over the world (Purushothaman et al., 2008). Seabuckthorn berries are an excellent source of phytochemicals such as ascorbic acid, tocopherols, unsaturated fatty acids, phenols, and carotenoids. The berries have been used for the treatment of radiation damage, burns, oral inflammation, and gastric ulcers (Zadernowskia et al., 2005). Earlier studies have reported the protective effects of the seed oil against liver injury, atopic dermatitis and atherosclerosis (Purushothaman et al., 2008). Other positive health effects that have been claimed include reduction of plasma cholesterol levels, inhibition of platelet aggregation, and regulation of immune function (Zadernowskia et al., 2005). In addition, seabuckthorn is uniquely capable of growing well under extreme environmental conditions such as water deficit, low temperature, salt stress, and high altitude (Xu et al., 2009). It can grow well in soil with a pH >9 and salt content >0.5%. In China, seabuckthorn has been used widely in the restoration of salt-alkaline grassland and forests. In this study, we compared the effects of salt stresses (9:1 molar ratio of NaCl to Na2 SO4 , pH 6.48–6.65) and alkali stresses (9:1 molar ratio of NaHCO3 to Na2 CO3 , pH 8.70–8.88) on the inorganic ion content and accumulation of organic acids in seabuckthorn to elucidate the mechanism by which plants adapt to alkali stress (high pH). 2. Materials and methods 2.1. Plant materials Seeds of seabuckthorn (H. rhamnoides L.) were collected from alkaline grassland in western Jilin Province, China. The seeds were sown in 17-cm diameter plastic pots that contained 2.5 kg of washed sand. The pots were watered sufficiently with Hoagland nutrient solution every 2 d. Distilled water was used to compensate for evaporation at other times. All pots were placed outdoors and protected from rain. Temperatures during the experimental period were 24–28 ◦ C during the day and 20–23 ◦ C at night. 2.2. Design of simulated salt and alkaline conditions Two neutral salts were mixed in a 9:1 molar ratio (NaCl:Na2 SO4 ), and applied to the salt stress group. For the alkali stress group, two alkaline salts were mixed in a 9:1 molar ratio (NaHCO3 :Na2 CO3 ). Within each group, three total salt concentrations were applied: 30, 60, and 90 mM. The salt stress groups were labeled S1–S3 and the alkali stress groups A1–A3. Therefore, in the 90 mM solution for salt stress, a mixture of 81 mM NaCl and 9 mM Na2 SO4 resulted in ion concentrations of 99 mM Na+ , 81 mM Cl− , and 9 mM SO4 2− . In the 90 mM solution for alkali stress, a mixture of 81 mM NaHCO3 and 9 mM Na2 CO3 resulted in ion concentrations of 99 mM Na+ , 81 mM HCO3 − , and 9 mM CO3 2− . In the salt stress and alkali stress groups, the pH was 6.48–6.65 and 8.70–8.88, respectively. 2.3. Stress treatments The stress treatments were applied when the seedlings were eight weeks old. Twenty-eight pots of uniformly growing seedlings were divided randomly into seven sets, four pots per set. Each pot was considered a single replicate. One set was used as an untreated
control, and the remaining six sets were treated with the various stress treatments. The pots were watered thoroughly daily at 17:00–18:00 h with nutrient solution that contained the appropriate stress salts. Control plants were maintained by watering with nutrient solution. The entire duration of treatment was 10 d. 2.4. Measurement of physiological indices Plants were harvested in the morning after the final treatment, and were first washed with tap water followed by distilled water. Roots, stems, and leaves were separated and freeze-dried. Dry samples of plant material (100 mg) were treated with 20 mL of deionized water at 100 ◦ C for 1 h, and the resulting extract was used to determine the contents of free inorganic ions and organic acids. The content of NO3 − , Cl− , SO4 2− , H2 PO4 − and oxalic acid was determined by ion chromatography using a DX-300 ion chromatographic system with an AS4A-SC ion-exchange column and a CDM-II electrical conductivity detector (mobile phase: Na2 CO3 /NaHCO3 = 1.7/1.8 mM; DIONEX, Sunnyvale, USA). The levels of other OAs were also determined by ion chromatography using the DX-300 ion chromatographic system with an ICE-AS6 ion-exclusion column, CDM-II electrical conductivity detector, and an AMMS-ICE II MicroMembrane suppressor (mobile phase: 0.4 mM heptafluorobutyric acid; DIONEX, Sunnyvale, USA). An atomic absorption spectrophotometer (TAS-990, Purkinje General, Beijing, China) was used to determine the levels of Na+ , K+ , Ca2+ , and Mg2+ . 2.5. Statistical data analysis Measurements were obtained from four replicates. Data were analyzed by one-way analysis of variance (ANOVA) using the statistical software SPSS 13.0. The mean values for the different treatments were compared by the post hoc least significant difference (LSD) test. Values of P < 0.05 were considered to indicate significance. 3. Results 3.1. Cations With increasing stress, the Na+ content of both leaves and stems increased (Fig. 1A and B, P < 0.05), and the increments under alkali stress were greater than those under salt stress. Neither increasing salt stress nor increasing alkali stress reduced the K+ content in H. rhamnoides leaves and stems; however, in roots, the K+ content decreased sharply with increasing alkali stress (Fig. 1D and E, P < 0.05). Alkali stress enhanced Ca2+ accumulation in leaves and stems, whereas salt stress reduced Ca2+ accumulation by a small amount (Fig. 1G–I, P < 0.05). The response of Mg2+ to both stresses was similar (Fig. 1J–L). With increasing salinity, the Mg2+ content in leaves and stems remained unchanged, whereas it decreased a little in roots. 3.2. Anions Under salt stress, the Cl− content increased with increasing salinity (P < 0.05; Fig. 2A–C). With increasing alkali stress, the Cl− content in leaves and stems remained unchanged, whereas it decreased a little in roots (P < 0.05; Fig. 2A–C). Salt stress affected the NO3 − content only slightly (Fig. 2). However, alkali stress, especially strong alkali stress, affected NO3 − accumulation clearly: the NO3 − content in leaves and stems increased but it decreased in roots (Fig. 2D–F). The H2 PO4 − content under alkali stress was lower than that under salt stress (Fig. 2G–I). Under salt stress, with increasing salinity, the SO4 2− content in the leaf and stem both remained relatively unchanged, whereas it increased slightly in the
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Fig. 1. Effects of salt and alkali stresses on contents of Na+ , K+ , Ca2+ , and Mg2+ in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 : Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05).
Table 1 Percentage contribution of seven organic acids to total moles of organic acid in H. rhamnoides L. leaves under salt and alkali stresses. Salinity (mM)
Malate (%)
Citrate (%)
Succinate (%)
Acetate (%)
Oxalate (%)
Formate (%)
Lactate (%)
Salt stress
Control 30 60 90
29.50 24.15 19.33 19.30
21.13 18.61 17.70 18.06
2.44 2.18 2.20 1.82
4.57 5.12 6.11 5.68
34.82 41.74 45.45 46.48
4.08 4.66 5.50 5.71
3.46 3.54 3.71 2.96
Alkali stress
30 60 90
35.16 33.32 37.62
18.39 17.06 19.18
2.63 2.51 2.48
4.52 4.21 2.48
33.43 33.49 30.10
3.48 3.42 2.89
2.38 5.99 5.25
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Table 2 Percentage contribution of seven organic acids to total moles of organic acid in H. rhamnoides L. stems under salt and alkali stresses. Salinity (mM)
Malate (%)
Citrate (%)
Succinate (%)
Acetate (%)
Oxalate (%)
Formate (%)
Lactate (%)
Salt stress
Control 30 60 90
26.73 19.76 19.31 16.99
15.94 16.19 15.63 15.68
0.85 0.87 0.76 0.73
8.77 11.76 11.11 11.17
41.59 44.52 47.22 48.99
4.19 4.53 4.09 3.95
1.95 2.37 1.89 2.50
Alkali stress
30 60 90
28.71 33.10 37.39
15.12 15.42 15.85
1.25 1.16 1.42
8.12 7.59 5.92
41.04 38.57 35.62
3.88 2.20 2.12
1.89 1.97 1.68
Table 3 Percentage contribution of seven organic acids to total moles of organic acid in H. rhamnoides L. roots under salt and alkali stresses. Salinity (mM)
Salt stress
Control 30 60 90
Alkali stress
30 60 90
Malate (%)
Citrate (%)
Succinate (%)
Acetate (%)
Oxalate (%)
Formate (%)
Lactate (%)
5.94 3.45 3.51 5.46
10.92 10.52 12.83 13.41
0 0 0 0
24.39 23.06 21.41 17.48
48.07 50.14 51.72 52.12
7.38 7.90 5.07 6.40
3.29 4.93 5.46 5.12
8.06 11.64 6.54
20.33 18.15 15.23
0 0 0
32.99 31.87 53.47
31.57 25.47 15.26
4.92 9.49 3.02
2.13 3.38 6.48
root (Fig. 2J–L). However, alkali stress increased the SO4 2− content in the leaf and decreased the SO4 2− content in the root, but did not affect SO4 2− accumulation in the stem (Fig. 2J–L). 3.3. Organic acids Malate, citrate, succinate, acetate, oxalate, formate, and lactate were detected in the leaves and stems of H. rhamnoides (Tables 1 and 2). In addition, malate, citrate, acetate, oxalate, formate, and lactate were detected in the root (Table 3). Salt stress affected the accumulation of OAs only slightly (Figs. 3 and 4), and even reduced OA accumulation in leaves (Fig. 4J). However, alkali stress had a large effect on OA accumulation. Alkali stress, especially strong alkali stress, clearly increased the levels of OAs in leaves and stems, but reduced OA accumulation in the root (Figs. 3 and 4). The levels of OAs in leaves and stems were much higher than those in the root (Figs. 3 and 4). Oxalate, malate, and citrate were the dominant OAs in leaves and stems (Tables 1 and 2). However, in roots, less malate and citrate were accumulated, whereas acetate accumulation was enhanced significantly (Fig. 3 and Table 3). Remarkably, strong alkali stress caused acetate to be the dominant component; it comprised 53.47% of the total OAs in roots (Fig. 4D–F and Table 3). 4. Discussion 4.1. Inorganic ions It has been reported that salt stress and alkali stress are actually two distinct types of stress and that the injurious effects of alkali stress on plants are more severe than those of salt stress (Yang et al., 2007, 2008a,b,c, 2009). Alkali stress involves the same stress factors as salt stress but with the added involvement of high pH. Adverse effects on roots are the basis of high pH stress (Yang et al., 2008a,b,c, 2009). The results reported here suggested that alkali stress (high pH) had a significant effect on the distribution and accumulation of inorganic ions in H. rhamnoides. Na+ is the main toxic ion in salinized soil. Low Na+ and high K+ in the cytoplasm are essential for the maintenance of a number of enzymatic processes (Munns and Tester, 2008). Na+ enters plant cells through the high-affinity K+ transporter (HKT) and through non-selective cation channels (Zhu, 2003). The similarity in size of the hydrated ionic radii of Na+ and K+ makes them difficult to discriminate between, and this is the basis of Na+ toxicity (Blumwald,
2000). Under salt stress, Na+ competes with K+ for uptake into roots (Munns, 2002; Munns and Tester, 2008). However, our results showed that neither increasing salt stress nor increasing alkali stress decreased the K+ content in H. rhamnoides leaves and stems; although, in roots, the K+ content decreased sharply with increasing alkali stress (Fig. 1). This phenomenon demonstrated that no competition occurred between Na+ and K+ absorption, which suggested that H. rhamnoides might have a unique pathway of Na+ absorption. Under alkali stress, the decreased K+ content in the root might be attributable to an inhibitory effect of high pH on K+ absorption, which relies on the transmembrane proton gradient (Munns, 2002; Munns and Tester, 2008). In addition, in leaves and stems, the increase in Na+ under alkali stress was much higher than that under salt stress (Fig. 1). We propose that this was not a response to osmotic stress, but a specific response to high pH stress. Alkali stress might weaken the controls on absorption or transport of Na+ , which in turn would lead to the sharp increase in Na+ content in leaves and stems (Fig. 1A), and disrupt the ionic balance or pH homeostasis in tissue. The increased level of Na+ in shoots under alkali stress might also be related to decreased exclusion of Na+ . It is well known that many plant species have a Na+ exclusion mechanism that depends on a Na+ /H+ antiporter, such as salt overly sensitive 1 (SOS1), which exchanges cytoplasmic Na+ for external H+ (Zhu, 2003; Munns and Tester, 2008). The exchange activity relies on the transmembrane proton gradient, which is established by H+ -ATPase (Zhu, 2003). Under strong alkali stress, the lack of external protons might weaken the exchange activity of the Na+ /H+ antiporter in the root plasma membrane (Munns and Tester, 2008), and possibly reduce exclusion of Na+ into the rhizosphere and enhance in vivo accumulation of Na+ . Neither alkali stress nor salt stress affected Mg2+ accumulation in H. rhamnoides. The response of free Ca2+ to each stress was interesting. Salt stress did not affect accumulation of Ca2+ in leaves and stems, but alkali stress did cause the Ca2+ content to increase (Fig. 1). However, in roots, both salt and alkali stress decreased the Ca2+ content (Fig. 1). This might be a strain-specific response of H. rhamnoides to alkali stress (high pH), whose unknown cause and function should be investigated further. Given that nitrogen is one of the most essential elements in all biological materials, changes in its nutrition and metabolism are of particular importance. NO3 − plays an important role in the maintenance of intracellular ionic balance and osmotic adjustment when plants are under salt or alkali stress (Yang et al., 2007, 2008b, 2009).
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Fig. 2. Effects of salt and alkali stresses on contents of Cl− , SO4 2− , NO3 − and H2 PO4 − in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 :Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05). Table 4 Percentage of the contribution of various free anions to total negative charge in H. rhamnoides L. tissue under salt and alkali stresses. Salinity (mM)
Salt stress
Control 30 60 90
Alkali stress
30 60 90
Leaf
Stem
Cl− (%)
SO4 2− (%)
NO3 − (%)
H2 PO4 − (%)
OA (%)
Cl− (%)
SO4 2− (%)
NO3 − (%)
H2 PO4 − (%)
OA (%)
10.07 17.95 22.16 22.88
8.72 8.80 8.81 9.22
1.71 1.54 1.16 1.00
8.73 8.09 8.72 8.34
70.77 63.61 59.14 58.57
10.22 15.62 19.39 22.00
18.65 15.61 14.20 14.05
2.18 1.59 1.51 1.12
13.88 14.26 14.51 14.71
55.07 52.92 50.39 48.11
7.53 6.81 6.29
7.55 9.45 8.25
1.19 1.50 2.34
5.28 4.41 3.91
78.46 77.83 79.22
8.71 7.92 7.44
12.29 10.56 9.46
1.46 2.51 4.36
10.32 8.46 8.49
67.22 70.55 70.25
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Fig. 3. Effects of salt and alkali stresses on contents of malate, citrate, formate and succinate in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 :Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05).
The effects of salt or alkali stress on NO3 − content in H. rhamnoides were interesting. Salt stress did not affect the accumulation of NO3 − , whereas it was affected strongly by alkali stress (Fig. 2). Under alkali stress, as salinity increased, the NO3 − content in both leaves and stems increased, but it decreased in roots (Fig. 2). Although we did not measure other physiological parameters involved in nitrogen metabolism, the changes in NO3 − content clearly indicated that alkali stress can interfere with the uptake or metabolism of NO3 − . We assumed that NO3 − was deoxidized in the root because the NO3 − content in the H. rhamnoides root was much higher than that in the shoot. It has been proposed that NO3 − uptake is mediated by a 2H+ /1NO3 − symport mechanism, which relies on the transmembrane proton gradient (Crawford and Glass, 1998). The reduction
in NO3 − in the root under alkali stress might be related to the lack of external protons due to the high pH, and should be investigated further. 4.2. Ionic balance and accumulation of organic acids Ionic imbalance in plants is caused mainly by the influx of excess Na+ (Munns and Tester, 2008; Yang et al., 2007). Plants in saline conditions usually accumulate inorganic anions such as Cl− , NO3 − , H2 PO4 − , and SO4 2− or organic anions to neutralize the high concentrations of cations and maintain a stable intracellular pH (Yang et al., 2007, 2008b). As shown in Table 4, different anions differed in their contribution to the overall negative charge. The percentage contri-
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Fig. 4. Effects of salt and alkali stresses on contents of lactate, acetate, oxalate, total organic acid (OA) in Hippophae rhamnoides. Eight-week-old seedlings were subjected to salt (NaCl:Na2 SO4 = 9:1; pH 6.48–6.65) or alkali (NaHCO3 :Na2 CO3 = 9:1; pH 8.70–8.88) stress for 10 d. The values are means (±SE) of four replicates. In each column, the data markers identified with the same letters are not significantly different (P < 0.05).
bution of various anions to the total negative charge ranged from high to low as follows: OAs, Cl− , SO4 2− , H2 PO3 − , NO3 − . Although OA was the dominant factor in maintaining ionic equilibrium in stressed H. rhamnoides, its percentage contribution to total negative charge under alkali stress was higher than under salt stress, and in addition was higher in leaves than in stems (Table 4). This implied that OA may play different roles during different types of stress or in different tissues. Under alkali stress, especially strong alkali stress, the synthesis of OAs was enhanced in H. rhamnoides to neutralize the massive influx of Na+ into leaves and stems (Fig. 1). Na+ might participate in the signal transduction pathway by which OA synthesis is induced during the response to alkali stress, and this should be investigated in future. During the response to alkali stress, different OAs had different roles in maintaining the intracellular ionic balance. This showed
that accumulation of OAs was not simply a passive response to a negative charge deficit, but was a result of active metabolic regulation after alkali stress had been sensed. The metabolic regulation of OAs under alkali stress may involve one or more key enzymes. These key enzymes may be known enzymes that participate in basal metabolic pathways such as the tricarboxylic acid cycle, glyoxylate cycle, glycolysis or other pathways. Enzyme activity can be regulated at the level of synthesis (transcription, translation and modification of new polypeptides), or after synthesis by the action of activators and inhibitors. If the pattern of OA accumulation in response to alkali stress can be defined clearly, it should be possible to identify and clone the key genes of plant alkali tolerance easily. This requires further investigation. In summary, OA accumulation is a central adaptive mechanism by which H. rhamnoides maintain intracellular ionic balance under
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alkali stress. H. rhamnoides accumulated three main OAs (oxalate, malate, and citrate) in leaves and stems (Tables 1 and 2). Comparing these results with those of our previous studies, the mechanism by which OA metabolism was modulated in H. rhamnoides was significantly different from that in Chenopodiaceae halophytes such as Kochia sieversiana (Yang et al., 2007) and Suaeda glauca (Yang et al., 2008b). In both the latter two species, the accumulated oxalate represents approximately 90% of total OA, under both alkali and salt stress. These data indicate that the modulation of OA metabolism may play different roles in different plant species. However, changes in OA metabolism are still an important in vivo adaptive response of halophytes to alkali stress, and should constitute an important future direction for research into the physiology of plant alkali stress. As mentioned previously, H. rhamnoides accumulated mainly malate, oxalate, and citrate in leaves and stems (Figs. 3 and 4, Tables 1 and 2). However, in roots, less malate and citrate accumulated, and acetate accumulation was enhanced significantly (Figs. 3 and 4, Table 3), which indicated that roots and shoots might use different mechanisms to modulate OA metabolism. As an alkali-tolerant plant, H. rhamnoides may have evolved unique mechanisms to regulate OA metabolism. Under alkali stress, especially strong alkali stress, H. rhamnoides may enhance the rate of anaerobic respiration, which would increase synthesis of acetate. Thus, H. rhamnoides may have a strong capacity to tolerate hypoxic conditions, which could contribute to its high alkali tolerance. It is well known that alkali stress can destroy soil structure and greatly decrease soil permeability, which can impose hypoxic stress. As a result of long-term adaptation to hypoxic conditions in its habitat (alkaline soil), H. rhamnoides may have evolved specific mechanisms to tolerate hypoxic stress. These should be investigated further. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 30671491) and the National Key Basic Research Program of China (No. 2007CB106800). We thank International Science Editing (ISE) from Ireland for language editing. References Blumwald, E., 2000. Sodium transport and salt tolerance in plants. Curr. Opin. Cell Biol. 12, 431–434. Campbell, S.A., Nishio, J.N., 2000. Iron deficiency studies of sugar beet using an improved sodium bicarbonate-buffered hydroponics growth system. J. Plant Nutr. 23, 741–757. Crawford, N.M., Glass, A.D.M., 1998. Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci. 3, 389–395. Gao, C., Wang, Y., Liu, G., Yang, C., Jiang, J., Li, H., 2008. Expression profiling of salinity–alkali stress responses by large-scale expressed sequence tag analysis in Tamarix hispid. Plant Mol. Biol. 66, 245–258. Hartung, W., Leport, L., Ratcliffe, R.G., Sauter, A., Duda, R., Turner, N.C., 2002. Abscisic acid concentration, root pH and anatomy do not explain growth differences
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