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. 2008 Oct 8;2(1):22–31. doi: 10.1093/mp/ssn058

Overexpression of SOS (Salt Overly Sensitive) Genes Increases Salt Tolerance in Transgenic Arabidopsis

Qing Yang a,b,c, Zhi-Zhong Chen a,b,c, Xiao-Feng Zhou a,b,c, Hai-Bo Yin a,b,c, Xia Li a,b,c, Xiu-Fang Xin a,b,c, Xu-Hui Hong a,b,c, Jian-Kang Zhu c,d, Zhizhong Gong a,b,c,1
PMCID: PMC2639737  PMID: 19529826

Abstract

Soil salinity is a major abiotic stress that decreases plant growth and productivity. Recently, it was reported that plants overexpressing AtNHX1 or SOS1 have significantly increased salt tolerance. To test whether overexpression of multiple genes can improve plant salt tolerance even more, we produced six different transgenic Arabidopsis plants that overexpress AtNHX1, SOS3, AtNHX1 + SOS3, SOS1, SOS2 + SOS3, or SOS1 + SOS2 + SOS3. Northern blot analyses confirmed the presence of high levels of the relevant gene transcripts in transgenic plants. Transgenic Arabidopsis plants overexpressing AtNHX1 alone did not present any significant increase in salt tolerance, contrary to earlier reports. We found that transgenic plants overexpressing SOS3 exhibit increased salt tolerance similar to plants overexpressing SOS1. Moreover, salt tolerance of transgenic plants overexpressing AtNHX1 + SOS3, SOS2 + SOS3, or SOS1 + SOS2 + SOS3, respectively, appeared similar to the tolerance of transgenic plants overexpressing either SOS1 or SOS3 alone.

Keywords: abiotic/environmental stress, salinity, signal transduction

INTRODUCTION

Soil salinity is a major factor that limits the yield of agricultural crops. Plants require several mineral nutrients for their growth and development. However, excessive soluble ions including sodium and chloride are harmful to most plants, including all major crops (Hasegawa et al., 2000; Zhu, 2001; Chinnusamy et al., 2006). High concentrations of salt cause both ionic stress and osmotic stress that, in turn, lead to secondary stresses such as oxidative stress and nutritional disorders (Hasegawa et al., 2000; Zhu, 2001; Chinnusamy et al., 2006). One of the main strategies for improving plant salt tolerance is through the overexpression of genes that are either induced by stress and/or have been shown to be required for normal levels of tolerance.

Overexpression of genes encoding Late Embryogenesis Abundant (LEA) proteins, which accumulate to high levels during seed development, such as the barley HVA1 (Xu et al., 1996) and wheat dehydrin DHN-5 (Brini et al., 2007), can enhance plant salt tolerance, although their function is obscure. Osmotic stress also induces the accumulation of osmolytes, which serve as osmoprotectants and function for osmotic adjustment. Transgenic plants overexpressing the genes participating in the synthesis or accumulation of these osmolytes, such as mannitol (Tarczynski et al., 1992), proline (Kishor et al., 1995), ononitol (Sheveleva et al., 1997), glycinebetaine (Holmstrom et al., 2000), trehalose (Garg et al., 2002; Jang et al., 2003), fructan (Pilon-Smits et al., 1995), ectoine (Nakayama et al., 2000), or sorbitol (Gao et al., 2001), show increased salt tolerance. Overexpression of regulatory genes in signalling pathways, such as transcription factors (DREB/CBF) (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Kasuga et al., 1999), and protein kinases (MAPK, CDPK) (Kovtun et al., 2000; Saijo et al., 2000; Moon et al., 2003; Teige et al., 2004), also increases plant salt tolerance. Other genes that encode enzymes that are involved in oxidative protection, such as glutathione S-transferase, peroxidase, superoxide dismutase, ascorbate peroxidases, and glutathione reductases, can also be modified to improve plant salt tolerance (Gupta et al., 1993; Roxas et al., 1997; Lee et al., 2007). Finally, enhanced salt tolerance can also be achieved by overexpression of several other genes that regulate ion homeostasis, such as SOS1 (Shi et al., 2003), AtNHX1 (Apse et al., 1999; Zhang and Blumwald, 2001; Zhang et al., 2001), and AVP1 (Gaxiola et al., 2001).

In Arabidopsis, ion homeostasis is mediated mainly by the SOS signal pathway, which consists of three main components. SOS1 encodes a plasma membrane Na+/H+ antiporter that plays a critical role in sodium extrusion and in controlling long-distance Na+ transport from the root to shoot (Shi et al., 2000, 2002). SOS3 encodes an EF-hand Ca2+-binding protein that functions as a calcium sensor for salt tolerance (Liu and Zhu, 1998). SOS2 encodes a Ser/Thr protein kinase (Liu et al., 2000). Salt stress elicits a transient increase of Ca2+ that is sensed by SOS3. SOS2 interacts with and is activated by SOS3 (Halfter et al., 2000). The SOS2/SOS3 kinase complex phosphorylates and activates SOS1 (Qiu et al., 2002). In yeast, co-expression of SOS1, SOS2, and SOS3 increased the salt tolerance of transformed yeast cells much more than expression of one or two SOS proteins (Quintero et al., 2002), suggesting that the full activity of SOS1 depends on the SOS2/SOS3 complex.

AtNHX1 is the first studied plant vacuolar protein that can mediate Na+ transport into vacuoles (Apse et al., 1999). Overexpression of SOS1 or AtNHX1 has been reported to improve the plant salt tolerance (Apse et al., 1999; Zhang and Blumwald, 2001; Zhang et al., 2001; Shi et al., 2003). Because SOS1 requires the SOS2/SOS3 complex for its maximal activity, and AtNHX1 activity has been shown to be controlled by the SOS pathway (Qiu et al., 2004), the full activities of both SOS1 or AtNHX1 in transgenic plants may require SOS2 and SOS3. We hypothesized that transgenic plants co-expressing SOS1, SOS2, and SOS3 together may improve salt tolerance more than transgenic plants overexpressing any of the single genes, since SOS3/SOS3 may be required to fully activate both SOS1 and AtNHX1.

Here, we provide evidence to show that overexpression of AtNHX1 in Arabidopsis did not improve plant salt tolerance in Arabidopsis significantly above that of control plants. The transgenic plants overexpressing SOS3 or SOS1 did exhibit a clear increase in salt tolerance compared to the control plants. However, salt tolerance in transgenic plants overexpressing SOS1 + SOS2 + SOS3 was improved only marginally compared to transgenic plants overexpressing only SOS3 or SOS1.

RESULTS

Northern Blot Analyses of Expression of SOS Genes and AtNHX1 in Transgenic Arabidopsis Plants

Transformation vectors containing SOS1, SOS3, SOS2 + SOS3, AtNHX1, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3 were constructed (Figure 1A). The expression of SOS1 and AtNHX1 was under control of the super promoter (Gong et al., 2002) and SOS2 and SOS3 was controlled by the 35S promoter. Each construct containing different gene(s) and an empty vector were introduced into Arabidopsis (Columbia gl1 background) by the floral dip method. Several independent transgenic lines were selected for each construct. To avoid gene silencing, we selected two stable single insert homozygous T4 lines from transgenic plants expressing each transgene(s) for further detailed studies. We used transgenic plants transformed with an empty vector as control. Northern blot analysis indicated that the transgenic lines expressed higher levels compared to control plants with or without salt treatment (Figure 1B–1G). Although we have observed that NaCl can increase the levels of accumulated SOS1 transcript, as reported previously (Shi et al., 2003), under the conditions used here, this was observed (Figure 1E).

Figure 1.

Figure 1.

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Vector Constructs and Northern Blot Analyses of Different Transgenic Lines under Normal and Salt Treatment Conditions.

Ten-day grown seedlings were transferred to MS medium containing 150 mM NaCl for 12 h, and total RNAs were extracted and used for Northern blots. Tubulin was used as a loading control.

(A) Vector structures used in this study.

(B) Northern blot of transgenic plants overexpressing AtNHX1.

(C) Northern blot of transgenic plants overexpressing SOS3.

(D) Northern blot of transgenic plants overexpressing AtNHX1 + SOS3.

(E) Northern blot of transgenic plants overexpressing SOS1.

(F) Northern blot of transgenic plants overexpressing SOS2 + SOS3.

(G) Northern blot of transgenic plants overexpressing SOS1 + SOS2 + SOS3.

Salt Tolerance Analysis of Transgenic Plants

To test whether co-expression of several salt-related genes could enhance salt tolerance in transgenic plants, we analyzed the response of transgenic plants to salt stress by a root bending assay (Zhu et al., 1998). Seedlings grown on MS medium for 4 d were transferred to new MS medium supplemented with 170, 200, or 220 mM NaCl, respectively, and cultured for three more days. Without salt treatment, all the transgenic plants showed no differences in growth compared to control plants. However, during the salt treatment, all of the transgenic plants showed better growth than the control plants, except for the transgenic plants overexpressing only AtNHX1 (Figure 2A). Under 170 mM NaCl treatment for 3 d, we did not observe any growth differences among vector only and different transgenic plants. Under 200 mM NaCl for 3 d, some cotyledons of wild-type and transgenic plants expressing only AtNHX1 became white, but the cotyledons of other transgenic plants still retained green. Under 220 mM NaCl treatment for 3 d, less than 20% of the control plants and transgenic plants overexpressing only AtNHX1 survived, but over 80% of the transgenic plants overexpressing SOS1, SOS3, SOS2 + SOS3, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3, respectively, survived (Figure 2A and 2B). It appears that transgenic plants overexpressing SOS1 + SOS2 + SOS3 showed slightly higher survival compared to others, but were not dramatically different from SOS1.

Figure 2.

Figure 2.

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Root Bending Assay of Transgenic Plants Grown on MS Medium Containing Different Concentrations of NaCl.

(A) Four-day seedlings grown on MS medium were transferred to MS solid medium containing 0, 170, 200, or 220 mM NaCl, respectively, and cultured for three more days, and then pictures were taken.

(B) Analysis of survival rate of the transgenic and control plants treated with under 220 mM NaCl for 3 d in three independent experiments. Fifteen seedlings were counted in each experiment. Data are means ± SE; * P < 0.05, ** P < 0.01.

In another salt tolerance assay, seedlings grown on MS medium for 6 d were transferred to MS liquid medium supplemented with 200 mM NaCl or no NaCl for 6 d in liquid cultural conditions. Most of the wild-type and transgenic plants overexpressing only AtNHX1 died under these conditions but most of the other transgenic plants survived (Figure 4A). In several experiments, we did not observe any increased salt tolerance in transgenic plants overexpressing only AtNHX1, in contrast to earlier reports (Apse et al., 1999). We continued to examine more independent transgenic lines overexpressing AtNHX1. We finally checked over 130 AtNHX1 transgenic lines, but did not find any line with increased salt tolerance (data not shown). Although plants overexpressing NHX1 + SOS3 were more tolerant than control plants, they were not more tolerant than plants overexpressing SOS3 alone.

Figure 4.

Figure 4.

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Salt Tolerance Assay and Total Chlorophyll Contents of transgenic plants.

(A) Salt tolerance assay of transgenic and control seedlings grown in MS liquid medium supplemented with 200 mM NaCl for 6 d.

(B) Total chlorophyll contents of transgenic plants and control plants treated with 200 mM NaCl. The data were obtained from three independent experiments. Data are means ± SE; * P < 0.05, ** P < 0.01.

Na+ and K+ Accumulation in Transgenic Plants

The maintenance of K+/ Na+ homeostasis is an important requirement for salt tolerance (Zhu, 2001). The apparent functions of the SOS signal components and of AtNHX1 are to maintain a low Na+ content and normal K+ content in the cytosol. The SOS pathway restricts net influx of Na+ ions whereas NHX1 is supposed to increase movement of Na+ from the cytosol to the vacuole (Apse et al., 1999). To test whether overexpression of these genes can reduce Na+ accumulation in plants, we compared the Na+ contents among the transgenic and control plants. The results indicate that the Na+ contents in transgenic plants overexpressing SOS1 or AtNHX1 were not different from those of the control plants with or without salt treatment. However, the transgenic plants overexpressing SOS3, SOS2 + SOS3, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3, respectively, accumulated less Na+ than the control plants under both normal and salt treatment conditions (Figure 3A).

Figure 3.

Figure 3.

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Na+ (A) and K+ (B) Accumulation in the Transgenic Plants and Control Plants.

Data were obtained from three independent experiments and are means ± SE; * P < 0.05, ** P < 0.01.

We did not find any differences in K+ contents between the transgenic plants overexpressing SOS1, AtNHX1 and control plants with or without salt treatment (Figure 3B). However, the transgenic plants overexpressing SOS3, SOS2 + SOS3, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3, respectively, accumulated the same level of K+ as the control plants under normal growth conditions, but more K+ than the control plants under salt treatment (Figure 3B).

Comparison of Chlorophyll Contents in Transgenic and Control Plants

Without salt treatment, no significant difference in total chlorophyll content was detected among the control and all of the different transgenic lines. However, salt treatment quickly decreased total chlorophyll content of the control plants and transgenic plants overexpressing AtNHX1. The extent of this decline was much less in the other transgenic lines (Figure 4B). These results indicated that the photosynthetic capacities of the transgenic plants overexpressing SOS1, SOS3, SOS2 + SOS3, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3, respectively, remained higher during salt treatment than those of the control plants and transgenic plants only overexpressing AtNHX1.

Overexpression of SOS Genes Alleviates the Salt Inhibition on Lateral Root Development

Salt stress also inhibits lateral root development. To test whether overexpression of these genes can alleviate this inhibition, we analyzed the response of lateral roots to salt stress. Seedlings grown on MS medium were transferred to the MS medium containing 0, 170, or 200 mM NaCl, respectively, and cultured for 7 d. Under normal conditions, the lateral root growth of all the transgenic lines showed no differences from that of control plants. After salt treatment, the lateral root development of both the transgenic plants and the control plants was inhibited, but the extent of this inhibition was less in the transgenic plants overexpressing SOS1, SOS3, SOS2 + SOS3, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3 compared to control plants and plants overexpressing only AtNHX1 (Figure 5A). We counted the number of lateral roots of the transgenic plants and control plants. The lateral root number of all the transgenic plants was not different from that of control plants without salt treatment. However, salt stress reduced the lateral root number less in the transgenic plants overexpressing SOS1, SOS3, SOS2 + SOS3, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3, respectively, than control plants (Figure 5B). There is no significant difference in lateral root number between control and AtNHX1-overexpressing plants under salt stress.

Figure 5.

Figure 5.

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Comparison of Lateral Root Development of Transgenic and control plants.

(A) Lateral root morphology of the transgenic and control seedlings grown on normal medium for 4 days and then treated with 0, 170, and 200 mM NaCl for 1 week.

(B) Number of lateral roots of transgenic plants and control plants treated with 0, 170, and 200 mM NaCl for 1 week. The root numbers were counted from 15 seedlings in each of three independent experiments. Data are means ± SE; * P < 0.05, ** P < 0.01.

DISCUSSION

SOS genes are the main known mediators of ion homeostasis during Na+ toxicity in Arabidopsis (Zhu, 2001). In this study, we obtained transgenic plants overexpressing more than one SOS gene and tested relative salt tolerance. Since the combination of SOS1 + SOS2 + SOS3 genes improved salt tolerance in yeast substantially over any single SOS gene, we unexpectedly did not observe greatly improved salt tolerance in transgenic Arabidopsis plants overexpressing SOS1 + SOS2 + SOS3. Since activated SOS1 (minus the C-terminus) results in a higher salt tolerance in yeast, that is similar to yeast with all three SOS genes expressed, only activation of the antiporter activity of SOS1 appears to be required to substantially elevate salt tolerance in yeast (Quintero et al., 2002). However, activation of SOS1 by inclusion of SOS2 and SOS3 in the transformants appears insufficient to produce strong salt tolerance in Arabidopsis. We also compared the salt tolerance of transgenic plants overexpressing only AtNHX1 or AtNHX1 + SOS3 together. In our assay conditions, we did not find any difference in salt tolerance between wild-type and transgenic plants overexpressing only AtNHX1, and the salt tolerance of plants overexpressing both SOS3 and AtNHX1 was not improved more as compared with plants overexpressing only SOS3.

Reconstitution of the SOS signaling pathway in yeast indicates that the full activity of SOS1 depends on both SOS2 and SOS3 (Quintero et al., 2002). The activity analysis for SOS1 in Arabidopsis also indicates that plasma membrane Na+/H+ exchange activity for SOS1 is regulated by SOS2 and SOS3 (Qiu et al., 2002). However, a recent study indicates that SOS3 is mainly expressed in roots, whereas, in shoots, SCABP8/CBL10, a SOS3 homolog, appears to be the main mediator for salt tolerance in Arabidopsis (Quan et al., 2007). SCABP8 also interacts with SOS2 to regulate the activity of SOS1 (Quan et al., 2007). scabp8 mutants are hypersensitive to salt in shoot tissues, but only exhibit moderate salt sensitivity in roots (Quan et al., 2007). However, SOS3 is not able to complement the salt-sensitive phenotype of scabp8 mutant, and overexpression of SCABP8 only partially rescues the salt-sensitive phenotype of sos3 mutants, suggesting the functions of SOS3 and SCABP8 are only partially overlapping, and cannot be reciprocally substituted in different cells. Furthermore, besides SOS2 and SOS3, there are 23 additional SOS2-like proteins (PKS/CIPKs) and six more SOS3-like proteins (CBLs/SCaBPs). Interactions among different SOS2-like and SOS3-like proteins constitute various combinations that may exhibit diverse influences on their target proteins (Guo et al., 2001; Kolukisaoglu et al., 2004). SOS2 regulates the activity of not only SOS1, but also vaculor cation/proton antiporters in the NHX family (Qiu et al., 2004) and the Ca2+/H+ exchanger CAX1 (Cheng et al., 2004). It is not unexpected that the complexes of SOS2- and SOS3-like proteins and their diverse targets may constitute a complex signaling network in different tissues and even in different cells (Guo et al., 2001; Kolukisaoglu et al., 2004). Transgenic plants overexpressing SOS3 showed the most increase in salt tolerance phenotype, suggesting that greater salt tolerance could be achieved by modulating upstream elements in the SOS signaling pathway. Because of results with yeast, it is usually assumed that increased salt tolerance results from greater antiporter activity of SOS1. However, we cannot exclude additional roles of SOS3 that could be independent of the known SOS pathway functions.

In this study, we did not observe a greatly increased salt tolerance in transgenic Arabidopsis plants overexpressing SOS1 + SOS2 + SOS3 compared to transgenic plants overexpressing either SOS1 or SOS3 alone. We speculate that because SOS3 functions mainly in roots, but not in shoots, even though SOS1 activity is more increased in roots of transgenic plants, they may not show more salt tolerance because SOS2 and SOS3 may not function optimally with SOS1 in shoots. It is also possible that salt tolerance in the whole plant needs other unidentified components that cooperate with SOS genes. Still another possibility is that the correct co-expression of SOS genes in certain cell types may be more important for improving Na+ extruding activity of SOS1 in roots. How Na+ is extruded out of roots is still not clearly understood, which may require different expression levels of SOS genes in different types of root cells. In the previous study, transgenic plants overexpressing SOS1 (driven by the 35S promoter) apparently reduced Na+ accumulation (Shi et al., 2003). However, Na+ levels in our transgenic plants overexpressing SOS1 did not change greatly compared with wild-type. Here, we used a constitutive super promoter to drive SOS1 expression. The super promoter may not match the temporal and spatial expression of native SOS1 very well, since the activity of the 35S promoter is so enhanced in the vasculature, which more closely matches the native SOS1 expression in parenchyma cells at the xylem–symplast boundary in Arabidopsis (Shi et al., 2002). The Na+ accumulation in transgenic plants overexpressing SOS3, AtNHX1 + SOS3, SOS2 + SOS3, or SOS1 + SOS2 + SOS3, respectively, was reduced when compared with that in wild-type plants under salt treatment, indicating that SOS1 antiporter activity can be increased by overexpression of SOS genes. However, because the expression of native SOS2 and SOS3 is unique and specific in some cell types, constitutive overexpression of SOS2 and SOS3 in other cells could also disturb coordinative functions of other proteins, resulting in a disturbance of normal function in salt tolerance.

Our results on transgenic plants overexpressing AtNHX1 suggest that AtNHX1 does not play a crucial role in salt tolerance in Arabidopsis. In order to confirm our initial results, we checked more than 100 independent transgenic lines with the root bending assay, and tested the salt tolerance of transgenic lines in soil watered with salt containing nutrient solution (data not shown). All of the assays failed to detect any increased salt tolerance in transgenic plants overexpressing only AtNHX1. We also tested the combination, or additive effect through comparing transgenic plants overexpressing AtNHX1 + SOS3 with transgenic plants overexpressing only SOS3, but found similar salt-tolerant phenotypes among these plants. Although we did not detect any additive effect of NHX1 overexpression when combined with SOS3, under different conditions, in which SOS3 is less effective, there could be a small additive effect. Our results are contrary to the previous report, in which the salt tolerance of transgenic Arabidopsis plants overexpressing AtNHX1 was greatly improved (Apse et al., 1999). Presently, we do not understand the reason for differences in the results (Apse et al., 1999). This could be due to different assay conditions or other unknown reasons. Recent results have indicated that NHX1,2 are primarily K+/H+ antiporters and may have modest effects on salt tolerance through the role in K+ transport (J. Pardo, personal communication). Research on AtNHX1 overexpression in Arabidopsis in other labs may be needed to re-examine the function of AtNHX1 in Arabidopsis.

METHODS

Plant Materials

Seeds were germinated and grown on MS medium supplemented with 3% (w/v) sucrose and 0.8% agar in a growth chamber with 24 h light at 22°C. Seedlings were grown in 340-ml pots filled with a mixture of peat/forest soil and vermiculite (3:1) in a greenhouse at 22°C, with light intensity of 50 μmol m−2 s−1 and 70% RH under long-day conditions (16 h light/8 h dark cycle).

Constructs and Plant Transformation

SOS1 or AtNHX1 cDNA was cloned as a XbaI–KpnI fragment downstream of the super promoter that consists of three copies of the octopine synthase enhancer in front of the manopine synthase promoter (Gong et al., 2002) in the pCAMBIA 1300 binary vector containing a hygromycin-resistant selectable marker. SOS3 cDNA was cloned as an NcoI–BstEII fragment downstream of the CaMV 35S promoter in the pCAMBIA 3301 binary vector containing a phosphinothricin-based herbicide-resistant selectable marker. SOS2 cDNA was cloned as an AscI–BamHI fragment downstream of the CaMV 35S promoter in the pGSA1276 binary vector containing a kanamycin-resistant selectable marker. AtNHX1 or SOS2 cDNA was inserted into the pCAMBIA 3301 binary vector containing the SOS3 cDNA by digestion with restriction enzymes HindIII and EcoRI. SOS1 cDNA was inserted into the pCAMBIA 3301 binary vector containing the SOS2 and SOS3 cDNA by digestion with restriction enzymes MluI and BstX1. The recombinant plasmids were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and then transferred into Arabidopsis thaliana (Columbia gl1 background) by the floral dip method. Transgenic plants harboring SOS1 or AtNHX1 were screened on MS agar medium containing 30 mg l−1 hygromycin. Transgenic plants harboring SOS3, SOS2 + SOS3, AtNHX1 + SOS3 or SOS1 + SOS2 + SOS3, respectively, were screened by treating plants with 519 μl l−1 Baster. T3 or T4 homozygous plants were used for salt-tolerant analysis.

RNA Hybridization

Seedlings grown on MS medium under continuous light for 10 d were transferred to MS solution containing 150 mM NaCl or no NaCl (for control) for 12 h. Total RNA was isolated and analyzed as previously described using different probes (Chen et al., 2005).

Analysis of Salt-Stress Tolerance

Four-day-old seedlings of transgenic plants and control plants were transferred to MS medium containing the indicated NaCl concentrations. After growing for the indicated time on the treatment medium, seedlings were photographed with a digital camera and the survival rates were measured and calculated. Growth was monitored using a root bending assay (Zhu et al., 1998).

Measurement of Na+ and K+ Contents

For Na+ and K+ content measurement in plant tissues, the 4-day-old Arabidopsis seedlings were transferred from MS medium to MS medium and MS medium containing 100 mM NaCl and treated for the indicated number of days. The plant tissues were treated in a muffle furnace at 575°C for 5 h and then dissolved in 0.1 N HCl. Na+ and K+ contents of the samples were measured by atomic absorption spectrophotometry (Xu et al., 2006).

Measurement of Chlorophyll Contents

Six-day-old seedlings grown on MS medium under continuous light were transferred to MS solution containing 200 mM NaCl or no NaCl (for control) for 6 d. Chlorophyll was extracted using 80% acetone and analyzed by using UV spectrophotometry.

Measurement of Lateral Root Number

Four-day-old Seedlings grown on MS medium under continuous light were transferred to MS medium containing the indicated NaCl concentrations. After growing for the indicated time (usually 7 d) on the treatment medium, seedlings were photographed with a digital camera and the numbers of lateral roots were measured and calculated.

FUNDING

This work was supported by grants from the National Natural Science Foundation of China to Z.G. (30370908 and 30421002). No conflict of interest declared.

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