Modulation of Ethylene Responses Affects Plant Salt-Stress Responses^,

Abstract

Ethylene signaling plays important roles in multiple aspects of plant growth and development. Its functions in abiotic stress responses remain largely unknown. Here, we report that alteration of ethylene signaling affected plant salt-stress responses. A type II ethylene receptor homolog gene NTHK1 (Nicotiana tabacum histidine kinase 1) from tobacco (N. tabacum) conferred salt sensitivity in NTHK1-transgenic Arabidopsis (Arabidopsis thaliana) plants as judged from the phenotypic change, the relative electrolyte leakage, and the relative root growth under salt stress. Ethylene precursor 1-aminocyclopropane-1-carboxylic acid suppressed the salt-sensitive phenotype. Analysis of Arabidopsis ethylene receptor gain-of-function mutants further suggests that receptor function may lead to salt-sensitive responses. Mutation of EIN2, a central component in ethylene signaling, also results in salt sensitivity, suggesting that EIN2-mediated signaling is beneficial for plant salt tolerance. Overexpression of the NTHK1 gene or the receptor gain-of-function activated expression of salt-responsive genes AtERF4 and Cor6.6. In addition, the transgene NTHK1 mRNA was accumulated under salt stress, suggesting a posttranscriptional regulatory mechanism. These findings imply that ethylene signaling may be required for plant salt tolerance.

Ethylene is a gaseous hormone that regulates plant growth and development. Based on the mutant analysis of triple responses of etiolated seedlings treated with ethylene, an ethylene signal transduction pathway has been proposed in Arabidopsis (Arabidopsis thaliana) that involves ethylene receptors, CTR1, EIN2, and EIN3, and other components (Bleecker and Kende, 2000; Wang et al., 2002; Chang and Bleecker, 2004; Guo and Ecker, 2004; Chen et al., 2005). Five receptor genes have been found in Arabidopsis, and they are classified into two subfamilies based on structural features. Subfamily I includes ETR1 and ERS1 (Chang et al., 1993; Hua et al., 1995). Subfamily II includes ETR2, EIN4, and ERS2 (Hua et al., 1998; Sakai et al., 1998). All these ethylene receptors can bind ethylene, and ETR1 has His kinase activity (Schaller and Bleecker, 1995; Gamble et al., 1998; Hall et al., 2000; O'Malley et al., 2005). Other Arabidopsis ethylene receptors had Ser kinase activity (Moussatche and Klee, 2004). The functions of the ETR1 kinase domain and its kinase activity are relatively weak in induction of ethylene response and seedling growth recovery after ethylene removal, and the signal output by the ETR N terminus is dependent on subfamily I members (Wang et al., 2003; Binder et al., 2004; Qu and Schaller, 2004; Xie et al., 2006). The ETR1 receptor function can be regulated by RTE1 or the copper transporter RAN1 (Hirayama et al., 1999; Resnick et al., 2006). ETR1 and ERS1 can interact with CTR1 that may be modulated by protein phosphatase 2A (Clark et al., 1998; Gao et al., 2003; Larsen and Cancel, 2003). Homologous genes of ethylene receptors have been isolated from many other plants, e.g. tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and rice (Oryza sativa; Cao et al., 2003). In tomato, six receptor genes have been cloned, and their functions in fruit ripening and defense response have been investigated (Ciardi et al., 2000, 2001; Tieman et al., 2000; Klee, 2002, 2004). In tobacco, four receptor genes have been isolated, and two of them, NTHK1 (N. tabacum histidine kinase 1) and NTHK2, represent subfamily II members (Knoester et al., 1997; Zhang et al., 1999a, 2001a, 2001b; Terajima et al., 2001). Both NTHK1 and NTHK2 have four hydrophobic regions, a GAF domain, a diverged His kinase domain, and a receiver domain. They share about 50% identity with the Arabidopsis subfamily II members EIN4 and ETR2. The biochemical property of NTHK1 and NTHK2 has been investigated, and NTHK1 had Ser/Thr kinase activity whereas NTHK2 had Ser/Thr and His kinase activity in the presence of Mn²⁺ and Ca²⁺, respectively (Xie et al., 2003; Zhang et al., 2004).

Ethylene has long been regarded as a stress hormone (Morgan and Drew, 1997). However, the roles of the ethylene signaling in abiotic stress responses remain an open question. Previously, we have cloned tobacco ethylene receptor genes NTHK1 and NTHK2, and studied their expression in response to different abiotic stresses. We found that both genes were induced by wounding and dehydration (Zhang et al., 1999a, 2001a, 2001b). In situ mRNA hybridization and immunohistochemistry analysis of the NTHK1 protein revealed that both mRNA and protein appeared first in the palisade cell layer upon cutting and then gradually spread to other sponge cells of the leaf (Zhang et al., 2001a; Xie et al., 2002). When exposed to salt stress, the NTHK1 gene expression was induced, whereas the NTHK2 expression was not significantly affected (Zhang et al., 2001a, 2001b). This differential expression pattern suggests different roles of various receptors in multiple stress responses and also implies a possible specific function for NTHK1 in plant salt-stress responses.

In this study, we generated transgenic Arabidopsis plants overexpressing the tobacco ethylene receptor homolog gene NTHK1 and investigated the plant responses to salt stress. The NTHK1 increased salt sensitivity of the transgenic plants and altered expression of salt-responsive genes. The Arabidopsis ethylene-response mutants were further examined for their response to salt stress. These studies have significance in elucidating the role of ethylene signaling in plant salt-stress response.

RESULTS

Phenotype and Ethylene Sensitivity of the NTHK1-Transgenic Plants

To investigate the function of tobacco ethylene receptor homolog gene NTHK1 in plant, we transformed the NTHK1 gene containing a 50-bp 5′-untranslated region (UTR), the open reading frame, and the 90-bp 3′-UTR sequences, driven by the 35S promoter, into Arabidopsis plants. It is assumed that similar downstream components existed in Arabidopsis as in tobacco plants since the Arabidopsis ethylene receptor gain-of-function mutant gene etr1-1 functioned in a tobacco background (Knoester et al., 1998). In total, 40 individual transgenic lines were obtained, and 90% of these lines showed large rosette and late flowering phenotype as exemplified by lines S1 and S10 (Fig. 1, A and B; data not shown). Homozygous T3 lines were recovered, and the two lines (S1 and S10, single insertion) were selected for further analysis. The rosette size of the two transgenic lines was comparable to that of the etr1-1 mutant (Fig. 1B). The large rosette of the two transgenic lines was most likely due to the enlargement of the epidermal cells on the leaf surface (Fig. 1C), whereas the late flowering probably resulted from the late development of the reproductive apex in the NTHK1-transgenic plants (Fig. 1C). It is not known if the mesophyll cells were also enlarged. The NTHK1 protein expression was confirmed in young leaves of the transgenic plants by immunohistochemical method (Fig. 1D).

Figure 1.

Phenotype and ethylene sensitivity of the NTHK1-transgenic plants. A, Phenotypic comparison of the wild-type Col-0, the NTHK1-transgenic lines S1 and S10, and the etr1-1 mutant. Plants have grown in pot for 4 weeks. B, Rosette sizes of the mature plants in A. Values represent means ± sd (n = 20). Differences for the transgenic lines and the etr1-1 compared with Col-0 are highly significant (P < 0.01). C, Scanning electron micrograph of the leaf surface and shoot apex from Col-0 and the transgenic line S10. a, Apex. Bars in the left sections are suitable for the right, with the first two bars representing 50 μm and the last bar 30 μm. D, NTHK1 protein distribution revealed by immunohistochemical analysis on cross-section of a developing leaf from 12-d-old seedlings of Col-0 or the transgenic line S10. Red color indicates positive signal. E, ACC dose-response curves of the seedling length for Col-0, the two transgenic lines S1 and S10, and the etr1-1 mutant. Values represent means ± sd (n = 25). F, ACC effect on Chitinase B gene expression in Col-0 and the transgenic line S10. Twelve-day-old seedlings were treated with 100 μm ACC and then subjected to RNA analysis with labeled NTHK1 and Chitinase B cDNA probes. G, Treatment with 2 μm AVG reduces the seedling length difference between wild type and the transgenic lines S1 and S10. Values represent means ± sd (n = 25). Differences between the transgenic lines and Col-0 are highly significant (P < 0.01). For B, E, and G, bars indicate sd.

NTHK1 is an ethylene receptor homolog gene, and the introduction of this gene may alter plant ethylene sensitivity. We then examined the ethylene sensitivity of the two NTHK1-transgenic lines S1 and S10. Upon 1-aminocyclopropane-1-carboxylic acid (ACC; precursor of the ethylene biosynthesis) treatment, the etiolated seedlings of the transgenic Arabidopsis showed reduced triple response when compared with the wild-type control (Fig. 1E), consistent with our previous observation in NTHK1-transgenic tobacco seedlings (Xie et al., 2002). The seedling length of the etr1-1 mutant was not significantly changed upon ACC treatment, in line with the ethylene insensitivity of this mutant (Fig. 1E). The expression of an ethylene-inducible gene, Chitinase B, was examined in both wild type and the transgenic plants. The results in Figure 1F show that this gene was inducible in wild-type plants upon ACC treatments, whereas it was not significantly affected in the transgenic line S10. The etiolated seedlings of the NTHK1-transgenic plants had longer hypocotyls, and ethylene biosynthesis blocker aminoethoxyvinylglycine (AVG)-treated wild type was comparable to the transgenic lines in seedling length (Fig. 1, E and G). All these results indicate that the NTHK1-overexpressing transgenic plants are less sensitive to ethylene.

NTHK1 Increases Salt Sensitivity of the Transgenic Plants and ACC Can Suppress This Sensitivity

Because the NTHK1 mRNA accumulation was observed in response to salt stress in tobacco seedlings (Zhang et al., 2001a), this gene may participate in salt-stress responses in plants. We tested the responses of the transgenic Arabidopsis under salt-stress conditions. Five-day-old seedlings were transferred onto the agar media containing various concentrations of NaCl and maintained for 7 d. It can be seen in Figure 2A that under normal condition, the transgenic plants appeared to be similar to wild type. However, in 50 mm NaCl, the transgenic lines exhibited slight epinasty phenotype, i.e. backward growth of the leaf blade and the petiole. This epinasty phenotype was very severe in the transgenic lines at 100 mm NaCl treatment. The petioles of the leaves in the transgenic lines were very short and the leaf blade grew backward close to base of the plant. The whole plant appeared to be very compact (approximately 0.5 cm in rosette size). More than 90% of the salt-stressed transgenic lines had this phenotypic change. The epinasty phenotype was in contrast with the relatively normal appearance (≥1.0 cm in rosette size) of the wild-type plants in the same plate of NaCl medium. It was also apparently different from the ethylene-caused symptom in Arabidopsis that has upward-erected petiole of light-green color and small leaf blade with curvature (Fig. 2B). At higher NaCl concentrations (150 and 200 mm), both wild type and the transgenic lines showed apparent growth inhibition. These results indicate that NTHK1-overexpressing lines are more sensitive to salt stress than the wild-type plants during this developmental stage.

Figure 2.

Phenotype of the NTHK1-transgenic plants and the ethylene-response mutants under salt stress. A, Comparison of the phenotype of the Col-0 and the transgenic lines S1 and S10 upon treatment with various concentrations of NaCl. Five-day-old seedlings of the Col-0 and the transgenic plants were transferred onto salt agar plates and maintained for 7 d to observe the phenotypic change. Please note the severe epinasty phenotype in 100 mm NaCl-treated S1 and S10 plants. B, Phenotype of ACC-treated Col-0 and the two transgenic lines S1 and S10. C, ACC suppression of the epinasty phenotype. The NTHK1-transgenic lines (S1 and S10) with the salt-induced epinasty phenotype were transferred onto 100 mm NaCl plus 10 μm or 100 μm ACC to observe the recovery of the epinasty phenotype. The 150 mm NaCl-treated wild type and the transgenic lines were also transferred onto NaCl plus ACC to observe phenotypic change. D, Comparison of the phenotype of Col-0, the transgenic plant S10, and the ethylene-response mutants etr1-1, ein4-1, ein2-1, and ein3-1 under NaCl or NaCl plus ACC. E, Comparison of the phenotype of the ethylene receptor loss-of-function mutants etr1-6, etr1-8, and ein4-7 and ethylene constitutive response mutant ctr1-1 under NaCl or NaCl plus ACC treatment.

Ethylene has been proposed to negatively regulate its receptor activity (Hua and Meyerowitz, 1998). It would suppress the salt-induced epinasty if this phenotype were the result of NTHK1 function. To test whether the epinasty phenotype in NTHK1-transgenic Arabidopsis can be altered by ethylene, the plants with the salt-induced epinasty were transferred to the salt medium with or without ethylene precursor ACC, and the phenotypic change was examined. ACC can be easily absorbed by plants and converted to ethylene. As shown in Figure 2C, when the S1 and S10 transgenic plants with the severe epinasty phenotype (Fig. 2A, 100 mm NaCl treatment) were still transferred onto 100 mm NaCl, the phenotype remained unchanged. However, when the transgenic plants with the same phenotype were transferred onto 100 mm NaCl plus ACC, they were all rescued rapidly from the second or the third day of the treatment and exhibited the phenotype resembling that of wild-type plants (Fig. 2C). The severe salt-stressed wild type and the transgenic plants from 150 mm NaCl treatment in Figure 2A were also transferred onto 150 mm NaCl plus ACC. The results showed that approximately 90% of the wild-type plants can be rescued, whereas approximately 50% to 70% of the transgenic plants can be fully or partially rescued (Fig. 2C, the most-right column). Other plant hormones cannot rescue the salt-induced epinasty phenotype (data not shown). These results likely indicate that the transgenic plants needed more ethylene to counteract the NTHK1 function that resulted in the salt-sensitive phenotype, and ethylene was required for salt-stress tolerance.

Comparison of the Phenotype of Arabidopsis Ethylene-Response Mutants under Salt Stress

Overexpression of the ethylene receptor homolog NTHK1 in transgenic plants appears to represent a gain of function in terms of ethylene signaling. Arabidopsis gain-of-function mutants of ethylene receptors, together with other ethylene-insensitive mutants in the ethylene-signaling pathway, were also tested for their responses in salt medium with or without ACC. The result in Figure 2D shows that all of the etr1-1 and ein4-1 gain-of-function mutant plants exhibited similar epinasty phenotype as the NTHK1-transgenic plants S10 had, and ein2-1plants, with a mutation in the membrane-localized EIN2 protein of the ethylene-signaling pathway (Alonso et al., 1999), also showed similar phenotype. However, ACC cannot rescue this phenotype in the three mutants, due to the ethylene-binding mutation (gain-of-function) in the etr1-1and ein4-1 genes, and the loss-of-function mutation in the ein2-1 gene. Under salt stress, ein3-1 mutant, with a mutation in the transcription factor EIN3 of the ethylene-signaling pathway (Chao et al., 1997), did not have the epinasty phenotype but showed wild type-like phenotype (Fig. 2D). These results probably indicate that the ethylene receptor and EIN2 are in the pathway regulating salt-induced phenotypic change, whereas EIN3 is not in this pathway.

We also tested if loss-of-function mutants of Arabidopsis ethylene receptors had any phenotypic change under salt stress. The results in Figure 2E show that none of the tested loss-of-function mutants, etr1-6, etr1-8, and ein4-7, had any epinasty phenotype upon salt stress as compared with the control plants under the same condition. These results further suggest that receptor function may lead to the salt-sensitive epinasty. ACC treatment did not significantly change the phenotype of the salt-treated loss-of-function mutants (Fig. 2E). The ethylene constitutive response mutant ctr1-1 did not show significant phenotypic alteration under either salt or ACC treatment in comparison with the Columbia (Col)-0 plants, except that the ctr1-1 mutants appeared to be slightly smaller than the Col-0 plants (Fig. 2E; data not shown).

Electrolyte Leakage in Salt-Stressed NTHK1-Transgenic Plants and Various Ethylene-Response Mutants

Relative electrolyte leakage represents an indicator for the damage caused by salt stress (Verslues et al., 2006). We measured the change of this parameter in salt-treated NTHK1-transgenic plants. Figure 3A shows that the two transgenic lines S1 and S10 had higher relative electrolyte leakage under treatment with 100 and 150 mm NaCl than the wild-type controls, indicating a susceptible response to salt stress in the transgenic plants. When the plants were treated with higher concentrations of NaCl, the relative electrolyte leakage tended to be comparable in both the NTHK1-transgenic plants and the wild-type controls (Fig. 3A). Because ACC can rescue the salt-induced epinasty phenotype, we tested if ACC can also alter the electrolyte leakage. Figure 3B show that ACC treatment significantly reduced the salt-induced increase of the relative electrolyte leakage by 26% and 31% in the two NTHK1-transgenic lines S1 and S10, respectively, but did not significantly affect this parameter in the Col-0 plants. Several ethylene-response mutants of Arabidopsis were also measured for their relative electrolyte leakage under salt stress (Fig. 3C). In comparison with the wild-type control, the etr1-1, ein2-1, ein4-1, and ein3-1 mutants, as well as the S1 and S10 plants, showed high leakage levels. The constitutive response mutant ctr1-1 and the three ethylene receptor loss-of-function mutants ein4-7, etr1-6, and etr1-8 all exhibited similar leakage levels to that of the Col-0 plants (Fig. 3C). The electrolyte leakage levels under normal condition were comparable among all the lines examined (Fig. 3C).

Figure 3.

Relative electrolyte leakage in the NTHK1-transgenic plants and the ethylene-response mutants. A, Comparison of the relative electrolyte leakage in Col-0 and the two NTHK1-transgenic lines S1 and S10 in response to the salt treatments of different NaCl concentrations. The treatment was performed in the same way as in Figure 2A. B, ACC effect on the salt-induced electrolyte leakage. The treatment was the same as in Figure 2C and 100 μm ACC was used. C, Comparison of the relative electrolyte leakage in various ethylene-response mutants after salt-stress treatment. Five-day-old seedlings were transferred onto 100 mm NaCl and maintained for 7 d. Plants were also transferred onto MS plates as controls (CK). The leaves were harvested for measurement. **, Differences for the mutants compared with wild-type Col-0 are highly significant (P < 0.01). *, Difference between etr1-1 and wild-type Col-0 is significant (P < 0.05). For A, B, and C, bars indicate sd. Each data point represents average from three independent experiments.

Relative Root Growth of the NTHK1-Transgenic Plants and Various Ethylene-Response Mutants under Salt Stress

The NTHK1-transgenic plants and the wild-type control plants were grown vertically on Murashige and Skoog (MS) or MS plus NaCl medium. The root length was measured and the ratios of the root length under salt stress to the root length under normal condition were calculated. These ratios represented the relative root length of the plants under salt stress and can be used to evaluate the root response to salt stress. Figure 4A shows that, from 75 mm to 150 mm NaCl treatment, the NTHK1-transgenic plants exhibited relatively short roots in comparison with the wild-type control, suggesting that NTHK1 confers salt-sensitive response to the root growth of the transgenic plants. Under treatments with other NaCl concentrations, no significant difference in relative root length was observed between wild type and the transgenic lines (Fig. 4A).

Figure 4.

Relative root growth of the NTHK1-transgenic plants and the ethylene-response mutants under salt stress. A, Relative root length of the wild type and the NTHK1-transgenic lines (S1 and S10) upon treatment with different concentrations of NaCl. B, Relative root length of various ethylene-response mutants under 100 mm NaCl treatment. Difference in comparison with wild-type plants is highly significant (**, P < 0.01) for ein4-1 and etr1-1. Difference in comparison with wild-type plants is significant (*, P < 0.05) for ein2-1. For A and B, bars indicate sd. The ratio of the average root length (from 20 seedlings) from salt plate to the average root length (from 20 seedlings) from MS plate was defined as relative root length. Each data point represents average of three independent experiments.

Various ethylene-response mutants were also examined for the relative root length under salt stress. The results in Figure 4B show that ein2-1, ein4-1, and etr1-1, similar to the NTHK1-transgenic lines (Fig. 4A; data not shown), had relatively short roots in comparison with the wild-type plants. However, the ein3-1 mutant and three ethylene receptor loss-of-function mutants, ein4-7, etr1-6, and etr1-8, did not show significant difference in this parameter when compared with the wild-type plants. The ctr1-1 mutant showed comparable root length to that of the wild-type plants (Fig. 4B). These results suggest that gain of function of the ethylene receptor or disruption of the EIN2 may lead to the inhibited root growth under salt stress, and EIN3 may not be in the pathway regulating the root growth under salt stress.

NTHK1 Regulates Expressions of Salt-Responsive Genes

Because the NTHK1-transgenic plants showed phenotypic and physiological changes under salt stress (Figs. 2, 3, and 4), we tested if NTHK1 regulated expressions of salt-responsive genes during the early stage of the salt stress. These genes included two transcription factor genes, AtERF4 (Fujimoto et al., 2000) and DREB2A (Liu et al., 1998), and four effector protein genes, Cor6.6, Erd10, rd17, and P5CS. The effector protein genes have been used as markers for stress-response studies (Cheong et al., 2003; Dubouzet et al., 2003). Figure 5A shows that the AtERF4 and Cor6.6 genes were up-regulated by approximately 2.5- to 5.3-fold in the two NTHK1-transgenic lines in comparison to their expression in Col-0 plants under normal condition. Under salt stress, whereas the AtERF4 and the Cor6.6 were induced by approximately 2.5- to 3.3-fold in Col-0 plants, their expression was superinduced by approximately 5- and 11-fold, respectively, in the transgenic lines compared with the nonstressed Col-0 plants. Slightly higher expressions of the rd17 gene in the two transgenic lines were also observed (Fig. 5A). For the DREB2A, Erd10, and P5CS genes, no significant difference in expression was observed between the two transgenic lines and the control plants, although these genes showed slight inductions under salt-stress condition, indicating that these genes were not regulated by the NTHK1 gene (Fig. 5A). It was interesting to find that the NTHK1 mRNA was accumulated under salt stress (Fig. 5A), and this phenomenon will be studied in the following text (Fig. 6).

Figure 5.

Expression of salt-responsive genes in the NTHK1-transgenic plants and the ethylene-response mutants upon NaCl treatment. A, Expression of six salt-responsive genes in Col-0 and the NTHK1-transgenic lines S10 and S1 upon treatment with water or 100 mm NaCl for 6 h. B, Expression of Cor6.6, rd17, and AtERF4 genes in etr1-1, ein4-1, and ein2-1 mutants upon treatment with 100 mm NaCl for the indicated times. C, NTHK1-inhibited gene expressions. Seedlings were subjected to treatment with water or 100 mm NaCl for 6 h and total RNA was isolated. BBC1, Lea, and AtNAC2 genes, identified from a microarray analysis, were used as probes for RNA-blot analysis.

Figure 6.

NTHK1 gene expression in the NTHK1-transgenic plants in response to salt and other treatments. A, NTHK1 expression in the S10 transgenic line after treatment with increasing concentrations of NaCl for 12 h. B, NTHK1 expression in the S10 transgenic line after treatment with 100 mm NaCl, or 100 mm NaCl plus 100 μm ACC. C, NTHK1 expression in the S10 transgenic line upon treatment with various abiotic stresses, chemicals, or different salts for 6 h. For all the sections, “rRNA” indicates the 18S rRNA gene.

The Arabidopsis ethylene-response mutants were also investigated for their effects on expression of the NTHK1-activated genes (Fig. 5B). Under normal condition, Cor6.6 expression was enhanced in etr1-1, ein4-1, and ein2-1 when compared with wild-type Col-0 plants, whereas rd17 expression was elevated in etr1-1 and ein4-1, but not in ein2-1. For AtERF4, its expression was in a similar level in all the four plants under normal condition. Under salt-stress condition, Cor6.6 expression was induced in all the plants tested, and the induction level was the strongest in etr1-1. For rd17 expression, the induction was stronger in etr1-1 and ein4-1 than that in ein2-1. For AtERF4 expression, the induction was earlier in etr1-1, ein4-1, and ein2-1 than that in Col-0 plants. These results indicate that gain-of-function mutation of the ethylene receptors ETR1 and EIN4 can enhance expression of Cor6.6, rd17, and AtERF4. EIN2 may be a negative regulator for Cor6.6 and AtERF4.

NTHK1 also down-regulated gene expressions. Figure 5C shows the expressions of three genes that were identified in a microarray analysis using salt-stressed Col-0 and salt-stressed NTHK1-transgenic plants (He et al., 2005). BBC1 (At3g49010) encodes 60S ribosomal protein L13 (RPL13B)/breast basic conserved protein 1-related. This gene expression was lowered by salt stress and further reduced in the NTHK1-transgencic line S10. On the contrary, the Lea and the AtNAC2 gene (He et al., 2005) were salt-inducible. However, the inductions of the two genes were reduced in intensity in the NTHK1-transgenic plants when subjected to salt treatment (Fig. 5C).

NTHK1 mRNA Accumulation in the Transgenic Plants under Various Treatments

The NTHK1 mRNA in the transgenic plants was accumulated under salt stress (Fig. 5A). We further investigated the mRNA level of this gene in the transgenic line S10 in response to salt and other treatments. It can be seen in Figure 6A that the NTHK1 mRNA was present in a relatively low level in the transgenic plants (S10). However, it was steadily accumulated upon treatment with increasing concentrations of NaCl. In wild-type plants, no signal was detected. Time-course study further demonstrated the induction of NTHK1 by salt stress, and ACC exerted no effect on the NTHK1 transcript level (Fig. 6B). The NTHK1-transgenic plants were also treated with other stresses and plant hormones or chemicals, and Figure 6C shows that the NTHK1 mRNA levels were not affected by treatments with polyethylene glycol, low temperature (4°C), heat shock (37°C), or wounding. Abscisic acid showed no influence on NTHK1 gene expression either. However, treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in dramatic accumulation of the NTHK1 transcripts (Fig. 6C), indicating that either de novo protein synthesis may not be required for the NTHK1 accumulation or a labile suppressor of NTHK1 expression requires de novo protein biosynthesis. The transgenic plants were also subjected to treatments with other salts, and Figure 6C shows that the NTHK1 accumulation was specifically induced by Na⁺ treatments but not significantly affected by K⁺, Li⁺, Cl⁻, and other anions tested.

DISCUSSION

Ethylene signaling is important in regulating plant growth and stress responses, and ethylene functions through its receptors. Although it is generally believed that ethylene signaling functions in multiple stress responses, it is not clear what specific roles the receptors can play under salt stress. In this study, we transformed a tobacco type II ethylene receptor homolog gene NTHK1 into Arabidopsis and found that the resulting transgenic plants, with NTHK1 mRNA and protein expression, were salt sensitive as can be seen from the severe epinasty phenotype, high electrolyte leakage, and reduced root growth under salt stress. Epinasty phenotype has been reported to correlate with the severity of salt stress (Jones and El-Beltagy, 1989; El-Iklil et al., 2000). Because overexpression of NTHK1 in the transgenic plants seems to represent gain of function of ethylene receptor, we speculate that the receptor function would lead to the salt-sensitive responses. The salt-sensitive epinasty phenotype and the high electrolyte leakage can be fully or partially suppressed by ACC treatment, suggesting that ethylene exerts a negative effect on ethylene receptors. This fact also suggests that ethylene may be required for plant salt tolerance. The gene expression ratios between the salt-stressed transgenic and salt-stressed Col-0 plants, identified by microarray analysis, can also be reversed by ACC to the normal ratios under MS condition (data not shown; Cao, 2004). This effect conferred by ACC application further confirmed the negative regulatory mechanism between ethylene and its receptors (Hua and Meyerowitz, 1998). In addition, ACC can rescue the salt-stressed phenotype in wild-type plants, further implying that ethylene signaling is beneficial for plant survival under salt stress.

The ethylene receptor gain-of-function mutants etr1-1 and ein4-1 exhibited salt sensitivity as observed from their severe epinasty phenotype, the high leakage levels, and reduced root growth, indicating that receptor functions may result in sensitive responses upon NaCl treatment. However, loss-of-function mutants ein4-7, etr1-6, and etr1-8 of these receptors do not have apparent improvement in salt tolerance in comparison with the wild-type plants, possibly implying that loss of a single receptor does not significantly affect the plant responses to salt stress. This fact may be consistent with the report that single loss-of-function receptor mutants did not have ethylene-response phenotype (Hua and Meyerowitz, 1998). However, an ETR1 single loss-of-function mutant etr1-7 has been found to have enhanced sensitivity and exaggerated response to ethylene (Cancel and Larsen, 2002). Whether this mutant has any tolerance to salt stress remains to be investigated.

The ethylene-signaling mutant ein2-1 also exhibited sensitivity under salt stress, indicating that EIN2 promotes plant salt tolerance. EIN2 is a membrane-associated protein and plays central roles in the ethylene-signaling pathway (Alonso et al., 1999). This protein is essential for the salt-induced expression of the AtNAC2 gene, which promotes lateral root formation (He et al., 2005). Another ethylene insensitive mutant, ein3-1, did not have the phenotypic change under salt stress in comparison with the wild-type plants, suggesting that EIN3, a transcription factor in the ethylene-signaling pathway (Chao et al., 1997), is not required for the plant phenotypic response to salt stress. The signaling pathway may have switched to a branch pathway before EIN3 and then led to the salt-induced phenotypic responses. Alternatively, other EIN3 homologs may mediate these salt-induced phenotypic changes. It should be noted that although EIN3 appears not to be involved in phenotypic changes under salt stress, it may still play some roles for salt tolerance in the physiological aspects as can be seen from the high electrolyte leakage in the salt-stressed ein3-1 mutant. Slightly lower survival rate has been observed in the ein3-1 mutant under high salinity stress when compared with the wild-type control, suggesting a role for EIN3 in salt tolerance (Achard et al., 2006). Mutation of the EIN3-degradation pathway components EBF1 and EBF2 (Guo and Ecker, 2003; Potuschak et al., 2003) also results in salt tolerance, and EIN3 may promote salt tolerance by enhancing DELLA function (Achard et al., 2006). In this study, we did not find a significant change in salt response in the ctr1-1 mutant treated with 100 mm NaCl, although salt tolerance is expected in this mutant according to its constitutive ethylene-response trait. However, under higher (200 mm) salinity, the ctr1-1 mutants exhibited higher survival rate in comparison with the wild-type plants (Achard et al., 2006), consistent with the speculation that ethylene signaling is important for plant salt tolerance. It is possible that severe salt stress could detect the survival difference between wild type and the ctr1-1 mutant. Although ctr1-1 is tolerant to high salt, the present ACC-treated wild type can only tolerate to 150 mm NaCl. The reason for the difference is not known. It is possible that the wild type needs to manage the whole situation to make a balanced decision for later generation, whereas the mutant may be only survived. This phenomenon remains to be further investigated.

Overexpression of the NTHK1 gene results in long seedlings and large rosettes in transgenic plants in comparison with the wild type. Question may arise as to whether these phenotypes were caused by ethylene insensitivity or were a consequence of cell enlargement caused by NTHK1. It is apparent that the etr1-1 mutant seedling length is more insensitive to ethylene than the NTHK1-transgenic plants (Fig. 1E; Zhou et al., 2006), whereas their rosette sizes are comparable. In the presence of ethylene biosynthesis blocker AVG, the transgenic seedlings showed similar length to the Col-0 seedlings, possibly suggesting that NTHK1 could result in reduced ethylene sensitivity. It is possible that NTHK1 could lead to the reduced ethylene sensitivity in seedling length but cause cell enlargement in rosette leaves, and the rosette size is not related to ethylene sensitivity. The ethylene emission was also measured in seedlings of the transgenic plants and the wild-type plants, and no significant difference was detected (data not shown), further implying that the longer seedling of the transgenic plants is due to the reduced ethylene sensitivity.

Although the NTHK1-transgenic plants showed reduced sensitivity to ethylene, they were still responsive to ethylene because the NTHK1 still had the normal ethylene-binding site. Therefore, ACC can rescue the salt-stressed phenotype through ethylene binding to the receptors. On the contrary, the etr1-1 mutant was completely insensitive to ethylene because of the mutation in the ethylene-binding site of ETR1 and ethylene cannot bind to the mutated ETR1. Therefore, ACC cannot rescue the salt-stressed etr1-1. The present results that overexpression of the ethylene receptor gene NTHK1 led to reduced ethylene sensitivity may be consistent with previous reports. Ciardi et al. (2000) found that tomato ethylene receptor NR overexpression reduced ethylene sensitivity in seedlings and mature plants. Consistently, transgenic plants with reduced LeETR4 gene expression displayed multiple symptoms of extreme ethylene sensitivity (Tieman et al., 2000).

Salt injury to plants can be estimated by the relative electrolyte leakage, and more injury would lead to higher level of relative electrolyte leakage. Electrolyte leakage allows assessment of the intactness of cell membranes, and more leakage indicates more damage of the membrane system (Verslues et al., 2006). The NTHK1-transgenic plants and the ethylene-response mutants etr1-1, ein4-1, and ein2-1 all had higher levels of the relative electrolyte leakage under salt stress, consistent with the salt sensitivity observed from the salt-induced epinasty and inhibited root growth. ACC can partially suppress the salt-caused electrolyte leakage in NTHK1-transgenic plants, indicating that ethylene is required for plant tolerance response to salt stress. It should be noted that the ein2-1 mutant had the highest level of relative electrolyte leakage under salt stress when compared with other mutants, suggesting that EIN2 is the most important component in signaling salt-tolerant responses. It should also be noted that the ein3-1 mutant did not exhibit salt-induced epinasty or inhibited root growth under salt stress but still had high level of electrolyte leakage. This fact suggests that the phenotypic change was separated from the electrolyte leakage. It is possible that the ethylene receptor and EIN2 can regulate both aspects, whereas EIN3, a downstream factor, can only control electrolyte leakage but not phenotypic change.

NTHK1 enhances expression of salt-responsive genes AtERF4, Cor6.6, and rd17, indicating its role in salt-stress response. Constitutive receptor signaling in Arabidopsis ethylene receptor gain-of-function mutants etr1-1 and ein4-1 also promotes expression of these genes. Recently, AtERF4 has been found to be a transcriptional repressor conferring ethylene insensitivity in its transgenic Arabidopsis plants, and the AtERF4-overexpressing plants are hypersensitive to sodium chloride (McGrath et al., 2005; Yang et al., 2005). This observation is consistent with our present findings. Therefore, gain of function of ethylene receptors may confer ethylene insensitivity and salt sensitivity at least partially through activation of the AtERF4 gene. In addition to the activation of some gene expressions, NTHK1 also down-regulates expressions of salt-induced or salt-inhibited genes (Fig. 5C). Previously, we have found that a NAC-type transcription factor gene, AtNAC2, was salt inducible and involved in lateral root development (He et al., 2005). The salt induction of the AtNAC2 gene was reduced in intensity in the NTHK1-transgenic plants and the ethylene-response mutants etr1-1 and ein2-1 (He et al., 2005). These results suggest that ethylene signaling can regulate salt-stress response by controlling multiple gene expressions.

The NTHK1 gene is salt inducible in tobacco plants (Zhang et al., 2001a). The NTHK1 transcripts were also accumulated in the NTHK1-transgenic lines under salt-stress condition. Recently, we have identified that the salt-responsive element was likely located in the transmembrane-coding region of the NTHK1 gene (Zhou et al., 2006). It is possible that salt stress induces a component that can stabilize the NTHK1 mRNA by binding to the specific sequence. Alternatively, salt stress may inhibit a transcription repressor that usually binds to the salt-responsive element under normal condition, resulting in the transcript accumulation under salt stress. Another possibility is that salt stress inhibited the mRNA-degradation enzymes. Treatment with protein synthesis inhibitor CHX resulted in an even drastic accumulation of NTHK1 mRNA, indicating that either NTHK1 transcript accumulation does not require de novo protein biosynthesis or a labile suppressor of NTHK1 expression requires de novo protein biosynthesis. Because the expression of NTHK1 is driven by the constitutive cauliflower mosaic virus 35S promoter, the NTHK1 transcript is more likely regulated posttranscriptionally by the latter mechanism. In animal cell systems, degradation of mature mRNA is a regulated process that determines gene expression. cis-Regulatory elements have been found in the 5′-UTR, the coding region, and the 3′-UTR. trans-Acting proteins have also been identified and can affect the mRNA stability by interacting with the cis-elements (Holcik and Liebhaber, 1997). In plants, the cis-element responsible for the CHX-induced SAUR mRNA accumulation was localized in the open reading frame of this gene (Li et al., 1994). A Na⁺/H⁺ antiporter SOS1 gene, driven by the 35S promoter, has also been observed to have increased transcript level in transgenic Arabidopsis upon NaCl treatment (Shi et al., 2003).

Salt-induced accumulation of the NTHK1 transcripts has also been observed in transgenic tobacco plants overexpressing NTHK1 (Cao et al., 2006), indicating a consistency with the case in the present transgenic Arabidopsis. It is not known why a gene that leads to a salt-sensitive phenotype is induced upon salt treatment. It is possible that salt stress-caused inhibition or slower growth of a plant is an adaptive feature that would make plants grow slowly under stress and thus save resources for later recovery after the stress has been removed or alleviated, as suggested by Zhu (2001). In the present case, the NTHK1 may first sense the salt stress and slow down the growth. At the same time, the NTHK1 can also activate expression of salt-responsive genes that are beneficial for plant survival under salt stress. Later, the accumulation of ethylene will inhibit the NTHK1 action and gradually recover the growth.

In addition to salt stress, ethylene receptor may also play roles in hydrogen peroxide signaling (Desikan et al., 2005). A CTR1-like protein kinase gene was also induced by salt stress (Shiozaki et al., 2005). The ein2-1 mutant has been found to be sensitive to heat stress and osmotic stress (Suzuki et al., 2005). Other His kinase genes may function as osmosensors in cyanobacterium, yeast, and Arabidopsis (Urao et al., 1999; Marin et al., 2003; Reiser et al., 2003). Plant cytokinin receptor Cre1, a His kinase, may also function as turgor sensor (Reiser et al., 2003).

Ethylene has been regarded as a stress hormone and is induced by many stresses (Abeles et al., 1992; Morgan and Drew, 1997). However, its role in salt stress is equivocal. El-Iklil et al. (2000) has reported that lower ethylene production was associated with salt tolerance. On the contrary, higher ethylene production has been regarded as an indicator for salt tolerance in rice (Khan et al., 1987). Ethylene has long been recognized as a growth inhibitor, but it can also promote growth (Pierik et al., 2006). A recent study suggests that ethylene signaling promotes salt tolerance in Arabidopsis (Achard et al., 2006). This study suggests that ethylene receptor function leads to salt sensitivity and ACC appears to suppress this salt sensitivity, implying that ethylene signaling is required for salt tolerance. Plant response to salt stress may depend on the balance and/or interaction between receptor and ethylene. When receptor signaling is prevalent, the plant is sensitive to salt stress but shows large rosette and late flowering. When ethylene signaling is prevalent, the plant is tolerant to salt stress but has small rosette and early flowering. The plant needs to make adjustment between these two extreme situations. Fine-tuning at various levels may make plants in an active homeostasis, and then the plants can survive better under stress condition and have relatively normal growth.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Seeds of Arabidopsis (Arabidopsis thaliana; ecotype Col-0), its ethylene-insensitive mutants etr1-1, ein2-1, ein4-1, and ein3-1, ethylene constitutive response mutant ctr1-1, and ethylene receptor loss-of-function mutants etr1-6, etr1-8, and ein4-1 were treated with 70% ethanol for 5 min and then sterilized with 15% bleach (Kao). After washing five times with sterile water, the seeds were plated on solidified MS medium (Murashige and Skoog, 1962). The seeds were stratified at 4°C for 2 d and then germinated at 23°C under continuous illumination condition. For pot growth, seeds were sown in pots (8 × 10 cm) containing vermiculite soaked with one-quarter-strength MS solution and then germinated in growth chambers at 23°C under continuous illumination.

Constructs and Arabidopsis Transformation

The full length of NTHK1 cDNA containing 45 bp of 5′-UTR and 79 bp of 3′-UTR was amplified from the original NTHK1 plasmid (Zhang et al., 2001a) and cloned into the BamHI and KpnI sites of the binary vector pBIN438 as described previously (Xie et al., 2002). The gene was driven by two copies of the 35S promoter. The tobacco mosaic virus Ω sequence was also included downstream of the 35S promoter to enhance the translation efficiency. The construct was introduced into Agrobacterium tumefaciens strain GV3101 and then transformed into Arabidopsis (Col-0) according to the vacuum infiltration method. The transgenic plants were screened on MS agar medium containing 50 mg/L Kanamycin. The presence and integration of the transgene was confirmed by Southern-blot analysis using the NTHK1 cDNA as a probe. T3 homozygous lines were used for further analysis.

Salt Stress and Other Treatments

Five-day-old seedlings from wild-type Arabidopsis (Col-0) and the NTHK1-transgenic lines were transferred onto MS medium containing 0, 50, 100, 150, and 200 mm NaCl. Each plate was divided into three or more equal regions to grow the Col and the transgenic seedlings. After around 7 d, the phenotypic change in these seedlings was observed. The NTHK1-transgenic lines showed epinasty phenotype at this stage in 100 mm NaCl when compared with the Col plants. These transgenic seedlings with the epinasty phenotype or the Col plants were further transferred onto MS medium with 100 mm NaCl or MS with NaCl and ACC (10 μm or 100 μm) to observe the rescue of the epinasty phenotype. Higher concentration of ACC facilitated rapid rescue of the phenotype. The ethylene-response mutants were also treated in the same way to compare the phenotypic change. The 150 mm NaCl-treated wild-type or transgenic plants were also transferred onto NaCl plus ACC to observe the phenotypic change.

To examine gene expression, 12-d-old seedlings of Arabidopsis Col-0, various NTHK1-transgenic lines, or the ethylene mutants were carefully pulled out from the plates and immersed in solution containing 100 mm or other concentrations of NaCl for various times. The NTHK1-transgenic seedlings of 12 d old were also immersed in 100 mm NaCl or 100 mm NaCl with 100 μm ACC for various times. The transgenic seedlings were treated in the same way for 6 h with 100 mm KCl, LiCl, Na₂SO₄, Na₂HPO₄, or Na₃ citrate to test their effect on the transgene expression. The NTHK1-transgenic seedlings were also immersed in solutions containing PEG8000 (10%, w/v), 100 μm abscisic acid originally dissolved in dimethyl sulfoxide (DMSO), 50 μm CHX originally dissolved in DMSO, DMSO (100 μL/50 mL solution), and 100 μm ACC for treatments. For wounding treatment, these seedlings were cut into slices and immersed in water. For 4°C and 37°C treatment, the seedlings were immersed in water and then placed at 4°C or 37°C. All the treatments above were performed in petri dishes containing 50 mL of water or various solutions for 6 h unless otherwise stated.

For triple-response test, the seeds were surface sterilized and stratified at 4°C for 2 d. The seeds were sown in MS plates containing various concentrations of ACC or 2 μm AVG (ethylene biosynthesis inhibitor). The plates containing the seeds were exposed to light for 8 h and then incubated in the dark for 4 d at 23°C. The total length of the seedlings, including the hypocotyls and roots, was measured and calculated. At least 25 seedlings were measured for each data point. The wild type, two NTHK1-transgenic lines S1 and S10, and the etr1-1 mutant were grown in pots, and the rosette size before bolting was measured for each plant.

For root growth under salt stress, seeds of wild-type plants, the NTHK1-transgenic lines, and various ethylene-response mutants were germinated vertically on MS or MS plus various concentrations of NaCl. For each NaCl treatment, three replicates were performed. After 9 d, the root length from 20 seedlings was measured for each replicate and average was calculated for each replicate. The ratio of the average root length from salt plate to the average root length from MS plate was calculated as relative root length. Three such ratios were further calculated for means. Three independent sets of experiments were performed and the averages were presented. The statistic analysis was performed using t test.

RNA Isolation and Northern-Blot Analysis

Total RNA isolation was performed following the description by Zhang et al. (1999b). Total RNA (20–30 μg) was fractionated on a 1.0% agarose gel containing formaldehyde, blotted onto nylon membranes, and hybridized as described previously (Zhang et al., 1999b). Probes were labeled with α-³²P-dCTP by the random-priming method. The exposure time for the NTHK1 gene in Figure 5C and the Cor6.6 gene in Figure 5B was longer than the exposure time for the corresponding genes in Figure 5A. All the northern analyses were repeated at least three times with independent RNA samples, and the results were consistent. Results from one set of the experiments were presented. Quantitation of the signal intensity was performed using the QuantiScan program.

The gene-specific DNA fragments were amplified by PCR, confirmed by sequencing, and used for probe labeling. The primers used were as follows: for Chitinase B, 5′-CAACGGTCTATGCTGCAG-3′ and 5′-ATATGAGCACTTGGATCC-3′; for AtERF4, 5′-CTATCCGAGAATGGCCAAG-3′ and 5′-AACAACATGGGGTGAAACC-3′; for Cor6.6, 5′-ACATCAAAAACGATTTTACAAG-3′ and 5′-GAACTTAAACTAGATTTTGTTG-3′; for Erd10, 5′-AGTTTCTCTTTATCATTCACG-3′ and 5′-AATAAAAGAGACAATGATCAAC-3′; for rd17, 5′-CTTAAAGCAACTACACAAGTC-3′ and 5′-ATCACAAAACACAGCGAATG-3′; and for P5CS, 5′-GACTAAGTTGACTCGTTCTC-3′ and 5′-CAACATCTAAATCATTCTCAG-3′. DREB2A plasmid was kindly provided by Dr. Q Liu (Tsinghua University, Beijing). Specific fragments for BBC1 (At3g49010), Lea (At2g41280), and AtNAC2 (At5g39610) were also amplified and used as probes for northern analysis.

Scanning Microscopy

For scanning electron microscopy, samples were first fixed in 2.5% glutaraldehyde for 4 h and then washed with phosphate buffer three times, each for 15 min. The materials were further fixed in 1% osmic acid (OsO₄) for 2 h and washed with phosphate buffer two times, each for 15 min. Samples were dehydrated in 30%, 50%, 70%, 85%, and 95% ethanol once for 20 min, and then in 100% ethanol for 15 min for two times. The samples were then treated with isopentyl acetate two times, each for 15 min. The materials were then dried using a critical point dryer (CPD030), gold coated using a sputter coater (SCD005), and examined under a scanning electron microscope (Hitachi S-570).

Immunohistochemical Analysis

The immunohistochemical analysis was performed following a previous report (Xie et al., 2002). The polyclonal antibody against the receiver domain of the NTHK1 protein (NTHK1-RD) was generated by immunizing rabbit with 2 mg of NTHK1-RD (Xie et al., 2002). The antiserum was purified using protein A-agarose (Roche) following the instructions. The specificity was proved by its non-cross-reactivity with the receiver domain of NTHK2. The sections were incubated overnight with the anti-NTHK1-RD antibody (1:1,000 dilution), and then with the alkaline phosphatase-conjugated goat anti-rabbit IgG (1:2,000 dilution; Sigma). Fast Red (Sigma) was used as a substrate, and red color revealed in cells or tissues represented a positive response. The sections were also stained with alcian blue for better observation of the morphology of the cells or tissues.

Measurement of Relative Electrolyte Leakage

Five-day-old seedlings from Col-0, two NTHK1-transgenic lines S1 and S10, and various ethylene-response mutants were transferred onto medium containing various concentrations of NaCl. After 7 d, the rosette leaves were harvested for measurement. The S1 and S10 plants with the epinasty phenotype were further transferred onto 100 mm NaCl plus ACC (100 μm) to observe the rescue of the epinasty. The rescued plants, together with proper controls, were also subjected to measurement. The plant leaves (0.1 g) were used to evaluate the electrolyte leakage by determining their relative conductivity in solution. The conductivity was determined using a conductivity detector DDS-11A (Kangyi). Briefly, the leaf segments from six to 10 seedlings were vacuum infiltrated in deionized water for 20 min and maintained in the water for 2 h. The conductivities (C1) of the obtained solutions were then determined. Then the leaf segments in deionized water were boiled for 15 min. After being thoroughly cooled to room temperature, the conductivities (C2) of the resulting solutions were determined. The values of C1 to C2 (C1/C2) were calculated and used to evaluate the relative electrolyte leakage. Each data point represents average from three independent experiments. The data were subjected to statistical analysis using t test.