Modulation of Ethylene Responses Affects Plant Salt-Stress Responses

Modulation of Ethylene Responses Affects PlantSalt-Stress Responses1[OA]

Wan-Hong Cao2, Jun Liu2, Xin-Jian He, Rui-Ling Mu, Hua-Lin Zhou, Shou-Yi Chen, and Jin-Song Zhang*

National Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academyof Sciences, Beijing 100101, China

Ethylene signaling plays important roles in multiple aspects of plant growth and development. Its functions in abiotic stressresponses remain largely unknown. Here, we report that alteration of ethylene signaling affected plant salt-stress responses. Atype II ethylene receptor homolog gene NTHK1 (Nicotiana tabacum histidine kinase 1) from tobacco (N. tabacum) conferred saltsensitivity in NTHK1-transgenic Arabidopsis (Arabidopsis thaliana) plants as judged from the phenotypic change, the relativeelectrolyte leakage, and the relative root growth under salt stress. Ethylene precursor 1-aminocyclopropane-1-carboxylic acidsuppressed the salt-sensitive phenotype. Analysis of Arabidopsis ethylene receptor gain-of-function mutants further suggeststhat receptor function may lead to salt-sensitive responses. Mutation of EIN2, a central component in ethylene signaling, alsoresults in salt sensitivity, suggesting that EIN2-mediated signaling is beneficial for plant salt tolerance. Overexpression of theNTHK1 gene or the receptor gain-of-function activated expression of salt-responsive genes AtERF4 and Cor6.6. In addition, thetransgene NTHK1 mRNA was accumulated under salt stress, suggesting a posttranscriptional regulatory mechanism. Thesefindings imply that ethylene signaling may be required for plant salt tolerance.

Ethylene is a gaseous hormone that regulates plantgrowth and development. Based on the mutant anal-ysis of triple responses of etiolated seedlings treatedwith ethylene, an ethylene signal transduction path-way has been proposed in Arabidopsis (Arabidopsisthaliana) that involves ethylene receptors, CTR1, EIN2,and EIN3, and other components (Bleecker and Kende,2000; Wang et al., 2002; Chang and Bleecker, 2004; Guoand Ecker, 2004; Chen et al., 2005). Five receptor geneshave been found in Arabidopsis, and they are classi-fied 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). Allthese ethylene receptors can bind ethylene, and ETR1has 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 Serkinase activity (Moussatche and Klee, 2004). The func-tions of the ETR1 kinase domain and its kinase activity

are relatively weak in induction of ethylene responseand seedling growth recovery after ethylene removal,and the signal output by the ETR N terminus is de-pendent 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 byRTE1 or the copper transporter RAN1 (Hirayama et al.,1999; Resnick et al., 2006). ETR1 and ERS1 can interactwith CTR1 that may be modulated by protein phos-phatase 2A (Clark et al., 1998; Gao et al., 2003; Larsenand Cancel, 2003). Homologous genes of ethylenereceptors have been isolated from many other plants,e.g. tomato (Solanum lycopersicum), tobacco (Nicotianatabacum), and rice (Oryza sativa; Cao et al., 2003). Intomato, six receptor genes have been cloned, and theirfunctions in fruit ripening and defense response havebeen investigated (Ciardi et al., 2000, 2001; Tiemanet al., 2000; Klee, 2002, 2004). In tobacco, four recep-tor genes have been isolated, and two of them, NTHK1(N. tabacum histidine kinase 1) and NTHK2, representsubfamily II members (Knoester et al., 1997; Zhanget al., 1999a, 2001a, 2001b; Terajima et al., 2001). BothNTHK1 and NTHK2 have four hydrophobic regions, aGAF domain, a diverged His kinase domain, and areceiver domain. They share about 50% identity withthe Arabidopsis subfamily II members EIN4 and ETR2.The biochemical property of NTHK1 and NTHK2 hasbeen investigated, and NTHK1 had Ser/Thr kinaseactivity whereas NTHK2 had Ser/Thr and His kinaseactivity in the presence of Mn21 and Ca21, respectively(Xie et al., 2003; Zhang et al., 2004).

Ethylene has long been regarded as a stress hor-mone (Morgan and Drew, 1997). However, the roles ofthe ethylene signaling in abiotic stress responses re-main an open question. Previously, we have cloned

1 This work was supported by the National Key Basic ResearchProject (grant no. 2006CB100102), the National High Tech Project(grant no. 2006AA10Z113), the Chinese Academy of Sciences (grantno. KSCXZ–YW–N–010), and the National Natural Science Founda-tion of China (grant nos. 30370130 and 30370132).

2 These authors contributed equally to the paper.* Corresponding author; e-mail [email protected]; fax 8610–

64873428.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instruction for Authors (www.plantphysiol.org) is:Jin-Song Zhang ([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.094292

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tobacco ethylene receptor genes NTHK1 and NTHK2,and studied their expression in response to differentabiotic stresses. We found that both genes were in-duced by wounding and dehydration (Zhang et al.,1999a, 2001a, 2001b). In situ mRNA hybridization andimmunohistochemistry analysis of the NTHK1 proteinrevealed that both mRNA and protein appeared firstin the palisade cell layer upon cutting and then grad-ually spread to other sponge cells of the leaf (Zhanget al., 2001a; Xie et al., 2002). When exposed to salt stress,the NTHK1 gene expression was induced, whereasthe NTHK2 expression was not significantly affected(Zhang et al., 2001a, 2001b). This differential expres-sion pattern suggests different roles of various recep-tors in multiple stress responses and also implies apossible specific function for NTHK1 in plant salt-stress responses.

In this study, we generated transgenic Arabidopsisplants overexpressing the tobacco ethylene receptorhomolog gene NTHK1 and investigated the plant re-sponses to salt stress. The NTHK1 increased salt sen-sitivity of the transgenic plants and altered expressionof salt-responsive genes. The Arabidopsis ethylene-response mutants were further examined for theirresponse to salt stress. These studies have significancein elucidating the role of ethylene signaling in plantsalt-stress response.

RESULTS

Phenotype and Ethylene Sensitivity of theNTHK1-Transgenic Plants

To investigate the function of tobacco ethylene re-ceptor homolog gene NTHK1 in plant, we transformedthe NTHK1 gene containing a 50-bp 5#-untranslatedregion (UTR), the open reading frame, and the 90-bp3#-UTR sequences, driven by the 35S promoter, intoArabidopsis plants. It is assumed that similar down-stream components existed in Arabidopsis as in to-bacco plants since the Arabidopsis ethylene receptorgain-of-function mutant gene etr1-1 functioned in atobacco background (Knoester et al., 1998). In total, 40individual transgenic lines were obtained, and 90% ofthese lines showed large rosette and late floweringphenotype as exemplified by lines S1 and S10 (Fig. 1, Aand B; data not shown). Homozygous T3 lines wererecovered, and the two lines (S1 and S10, single inser-tion) were selected for further analysis. The rosette sizeof the two transgenic lines was comparable to that ofthe etr1-1 mutant (Fig. 1B). The large rosette of the twotransgenic lines was most likely due to the enlargementof the epidermal cells on the leaf surface (Fig. 1C),whereas the late flowering probably resulted from thelate development of the reproductive apex in the NTHK1-transgenic plants (Fig. 1C). It is not known if the meso-phyll cells were also enlarged. The NTHK1 proteinexpression was confirmed in young leaves of the trans-genic plants by immunohistochemical method (Fig. 1D).

NTHK1 is an ethylene receptor homolog gene, andthe introduction of this gene may alter plant ethylenesensitivity. We then examined the ethylene sensitivityof the two NTHK1-transgenic lines S1 and S10. Upon1-aminocyclopropane-1-carboxylic acid (ACC; precur-sor of the ethylene biosynthesis) treatment, the etio-lated seedlings of the transgenic Arabidopsis showedreduced triple response when compared with the wild-type control (Fig. 1E), consistent with our previousobservation in NTHK1-transgenic tobacco seedlings(Xie et al., 2002). The seedling length of the etr1-1mutant was not significantly changed upon ACC treat-ment, in line with the ethylene insensitivity of this mu-tant (Fig. 1E). The expression of an ethylene-induciblegene, Chitinase B, was examined in both wild type andthe transgenic plants. The results in Figure 1F showthat this gene was inducible in wild-type plants uponACC treatments, whereas it was not significantly af-fected in the transgenic line S10. The etiolated seedlingsof the NTHK1-transgenic plants had longer hypocotyls,and ethylene biosynthesis blocker aminoethoxyvinyl-glycine (AVG)-treated wild type was comparable to thetransgenic lines in seedling length (Fig. 1, E and G). Allthese results indicate that the NTHK1-overexpressingtransgenic 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 ob-served 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 ofthe transgenic Arabidopsis under salt-stress condi-tions. Five-day-old seedlings were transferred onto theagar media containing various concentrations of NaCland maintained for 7 d. It can be seen in Figure 2A thatunder normal condition, the transgenic plants ap-peared to be similar to wild type. However, in 50 mM

NaCl, the transgenic lines exhibited slight epinastyphenotype, i.e. backward growth of the leaf blade andthe petiole. This epinasty phenotype was very severein the transgenic lines at 100 mM NaCl treatment. Thepetioles of the leaves in the transgenic lines were veryshort and the leaf blade grew backward close to base ofthe plant. The whole plant appeared to be very com-pact (approximately 0.5 cm in rosette size). More than90% of the salt-stressed transgenic lines had this phe-notypic change. The epinasty phenotype was in con-trast with the relatively normal appearance ($1.0 cmin rosette size) of the wild-type plants in the same plateof NaCl medium. It was also apparently different fromthe ethylene-caused symptom in Arabidopsis that hasupward-erected petiole of light-green color and smallleaf blade with curvature (Fig. 2B). At higher NaClconcentrations (150 and 200 mM), both wild type andthe transgenic lines showed apparent growth inhibi-tion. These results indicate that NTHK1-overexpressinglines are more sensitive to salt stress than the wild-type plants during this developmental stage.

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Ethylene has been proposed to negatively regulateits receptor activity (Hua and Meyerowitz, 1998). Itwould suppress the salt-induced epinasty if this phe-notype were the result of NTHK1 function. To testwhether the epinasty phenotype in NTHK1-transgenicArabidopsis can be altered by ethylene, the plants withthe salt-induced epinasty were transferred to the saltmedium with or without ethylene precursor ACC, andthe phenotypic change was examined. ACC can beeasily absorbed by plants and converted to ethylene.As shown in Figure 2C, when the S1 and S10 trans-genic plants with the severe epinasty phenotype (Fig.2A, 100 mM NaCl treatment) were still transferred onto100 mM NaCl, the phenotype remained unchanged.However, when the transgenic plants with the samephenotype were transferred onto 100 mM NaCl plus

ACC, they were all rescued rapidly from the second orthe third day of the treatment and exhibited thephenotype resembling that of wild-type plants (Fig.2C). The severe salt-stressed wild type and the trans-genic plants from 150 mM NaCl treatment in Figure 2Awere also transferred onto 150 mM NaCl plus ACC.The results showed that approximately 90% of thewild-type plants can be rescued, whereas approxi-mately 50% to 70% of the transgenic plants can be fullyor partially rescued (Fig. 2C, the most-right column).Other plant hormones cannot rescue the salt-inducedepinasty phenotype (data not shown). These resultslikely indicate that the transgenic plants needed moreethylene to counteract the NTHK1 function that re-sulted in the salt-sensitive phenotype, and ethylenewas required for salt-stress tolerance.

Figure 1. Phenotype and ethylene sensitivity of theNTHK1-transgenic plants. A, Phenotypic comparisonof the wild-type Col-0, the NTHK1-transgenic linesS1 and S10, and the etr1-1 mutant. Plants have grownin pot for 4 weeks. B, Rosette sizes of the matureplants in A. Values represent means 6 SD (n 5 20).Differences for the transgenic lines and the etr1-1compared with Col-0 are highly significant (P ,

0.01). C, Scanning electron micrograph of the leafsurface and shoot apex from Col-0 and the transgenicline S10. a, Apex. Bars in the left sections are suitablefor the right, with the first two bars representing50 mm and the last bar 30 mm. D, NTHK1 proteindistribution revealed by immunohistochemical anal-ysis on cross-section of a developing leaf from 12-d-old seedlings of Col-0 or the transgenic line S10. Redcolor indicates positive signal. E, ACC dose-responsecurves of the seedling length for Col-0, the twotransgenic lines S1 and S10, and the etr1-1 mutant.Values represent means 6 SD (n 5 25). F, ACC effecton Chitinase B gene expression in Col-0 and thetransgenic line S10. Twelve-day-old seedlings weretreated with 100 mM ACC and then subjected to RNAanalysis with labeled NTHK1 and Chitinase B cDNAprobes. G, Treatment with 2 mM AVG reduces theseedling length difference between wild type and thetransgenic lines S1 and S10. Values represent means6 SD (n 5 25). Differences between the transgeniclines and Col-0 are highly significant (P , 0.01). ForB, E, and G, bars indicate SD.

Ethylene Signaling and Salt-Stress Response

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Comparison of the Phenotype of Arabidopsis

Ethylene-Response Mutants under Salt Stress

Overexpression of the ethylene receptor homologNTHK1 in transgenic plants appears to represent again of function in terms of ethylene signaling. Arabi-dopsis gain-of-function mutants of ethylene receptors,together with other ethylene-insensitive mutants inthe ethylene-signaling pathway, were also tested fortheir responses in salt medium with or without ACC.The result in Figure 2D shows that all of the etr1-1 andein4-1 gain-of-function mutant plants exhibited similarepinasty phenotype as the NTHK1-transgenic plantsS10 had, and ein2-1plants, with a mutation in themembrane-localized EIN2 protein of the ethylene-

signaling pathway (Alonso et al., 1999), also showedsimilar phenotype. However, ACC cannot rescue thisphenotype in the three mutants, due to the ethylene-binding mutation (gain-of-function) in the etr1-1andein4-1 genes, and the loss-of-function mutation in theein2-1 gene. Under salt stress, ein3-1 mutant, with a mu-tation in the transcription factor EIN3 of the ethylene-signaling pathway (Chao et al., 1997), did not have theepinasty phenotype but showed wild type-like phe-notype (Fig. 2D). These results probably indicate thatthe ethylene receptor and EIN2 are in the pathwayregulating salt-induced phenotypic change, whereasEIN3 is not in this pathway.

We also tested if loss-of-function mutants of Arab-idopsis ethylene receptors had any phenotypic change

Figure 2. Phenotype of the NTHK1-transgenic plantsand the ethylene-response mutants under salt stress.A, Comparison of the phenotype of the Col-0 and thetransgenic lines S1 and S10 upon treatment withvarious concentrations of NaCl. Five-day-old seed-lings of the Col-0 and the transgenic plants weretransferred onto salt agar plates and maintained for7 d to observe the phenotypic change. Please note thesevere epinasty phenotype in 100 mM NaCl-treatedS1 and S10 plants. B, Phenotype of ACC-treatedCol-0 and the two transgenic lines S1 and S10. C,ACC suppression of the epinasty phenotype. TheNTHK1-transgenic lines (S1 and S10) with the salt-induced epinasty phenotype were transferred onto100 mM NaCl plus 10 mM or 100 mM ACC to observethe recovery of the epinasty phenotype. The 150 mM

NaCl-treated wild type and the transgenic lines werealso transferred onto NaCl plus ACC to observephenotypic change. D, Comparison of the phenotypeof Col-0, the transgenic plant S10, and the ethylene-response mutants etr1-1, ein4-1, ein2-1, and ein3-1under NaCl or NaCl plus ACC. E, Comparison of thephenotype of the ethylene receptor loss-of-functionmutants etr1-6, etr1-8, and ein4-7 and ethylene con-stitutive response mutant ctr1-1 under NaCl or NaClplus ACC treatment.

Cao et al.





under salt stress. The results in Figure 2E show thatnone of the tested loss-of-function mutants, etr1-6,etr1-8, and ein4-7, had any epinasty phenotype uponsalt stress as compared with the control plants underthe same condition. These results further suggest thatreceptor function may lead to the salt-sensitive epi-nasty. ACC treatment did not significantly change thephenotype of the salt-treated loss-of-function mutants(Fig. 2E). The ethylene constitutive response mutantctr1-1 did not show significant phenotypic alterationunder either salt or ACC treatment in comparison withthe Columbia (Col)-0 plants, except that the ctr1-1 mu-tants appeared to be slightly smaller than the Col-0plants (Fig. 2E; data not shown).

Electrolyte Leakage in Salt-Stressed NTHK1-Transgenic

Plants and Various Ethylene-Response Mutants

Relative electrolyte leakage represents an indicatorfor the damage caused by salt stress (Verslues et al.,2006). We measured the change of this parameter insalt-treated NTHK1-transgenic plants. Figure 3Ashows that the two transgenic lines S1 and S10 hadhigher relative electrolyte leakage under treatmentwith 100 and 150 mM NaCl than the wild-type controls,indicating a susceptible response to salt stress in thetransgenic plants. When the plants were treated withhigher concentrations of NaCl, the relative electrolyteleakage tended to be comparable in both the NTHK1-transgenic plants and the wild-type controls (Fig. 3A).Because ACC can rescue the salt-induced epinastyphenotype, we tested if ACC can also alter the elec-trolyte leakage. Figure 3B show that ACC treatmentsignificantly reduced the salt-induced increase of therelative electrolyte leakage by 26% and 31% in the twoNTHK1-transgenic lines S1 and S10, respectively, butdid not significantly affect this parameter in the Col-0plants. Several ethylene-response mutants of Arabi-dopsis were also measured for their relative electrolyteleakage under salt stress (Fig. 3C). In comparison withthe wild-type control, the etr1-1, ein2-1, ein4-1, andein3-1 mutants, as well as the S1 and S10 plants,showed high leakage levels. The constitutive responsemutant ctr1-1 and the three ethylene receptor loss-of-function mutants ein4-7, etr1-6, and etr1-8 all exhibitedsimilar leakage levels to that of the Col-0 plants (Fig.3C). The electrolyte leakage levels under normal con-dition were comparable among all the lines examined(Fig. 3C).

Relative Root Growth of the NTHK1-Transgenic Plantsand Various Ethylene-Response Mutants underSalt Stress

The NTHK1-transgenic plants and the wild-typecontrol plants were grown vertically on Murashigeand Skoog (MS) or MS plus NaCl medium. The rootlength was measured and the ratios of the root lengthunder salt stress to the root length under normalcondition were calculated. These ratios represented

the relative root length of the plants under salt stressand can be used to evaluate the root response to saltstress. Figure 4A shows that, from 75 mM to 150 mM

NaCl treatment, the NTHK1-transgenic plants ex-hibited relatively short roots in comparison with the

Figure 3. Relative electrolyte leakage in the NTHK1-transgenic plantsand the ethylene-response mutants. A, Comparison of the relativeelectrolyte leakage in Col-0 and the two NTHK1-transgenic lines S1and S10 in response to the salt treatments of different NaCl concen-trations. The treatment was performed in the same way as in Figure 2A.B, ACC effect on the salt-induced electrolyte leakage. The treatmentwas the same as in Figure 2C and 100 mM ACC was used. C, Compari-son of the relative electrolyte leakage in various ethylene-responsemutants after salt-stress treatment. Five-day-old seedlings were trans-ferred onto 100 mM NaCl and maintained for 7 d. Plants were alsotransferred onto MS plates as controls (CK). The leaves were harvestedfor measurement. **, Differences for the mutants compared with wild-type Col-0 are highly significant (P , 0.01). *, Difference betweenetr1-1 and wild-type Col-0 is significant (P , 0.05). For A, B, and C, barsindicate SD. Each data point represents average from three independentexperiments.






wild-type control, suggesting that NTHK1 conferssalt-sensitive response to the root growth of the trans-genic plants. Under treatments with other NaCl con-centrations, no significant difference in relative rootlength was observed between wild type and the trans-genic lines (Fig. 4A).

Various ethylene-response mutants were also exam-ined for the relative root length under salt stress. Theresults in Figure 4B show that ein2-1, ein4-1, and etr1-1,similar to the NTHK1-transgenic lines (Fig. 4A; datanot shown), had relatively short roots in comparisonwith the wild-type plants. However, the ein3-1 mutantand three ethylene receptor loss-of-function mutants,ein4-7, etr1-6, and etr1-8, did not show significantdifference in this parameter when compared with thewild-type plants. The ctr1-1 mutant showed compara-ble root length to that of the wild-type plants (Fig. 4B).These results suggest that gain of function of theethylene receptor or disruption of the EIN2 may lead

to the inhibited root growth under salt stress, andEIN3 may not be in the pathway regulating the rootgrowth under salt stress.

NTHK1 Regulates Expressions of Salt-Responsive Genes

Because the NTHK1-transgenic plants showed phe-notypic and physiological changes under salt stress(Figs. 2, 3, and 4), we tested if NTHK1 regulated ex-pressions of salt-responsive genes during the earlystage of the salt stress. These genes included two tran-scription factor genes, AtERF4 (Fujimoto et al., 2000)and DREB2A (Liu et al., 1998), and four effector pro-tein genes, Cor6.6, Erd10, rd17, and P5CS. The effectorprotein 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 Cor6.6genes were up-regulated by approximately 2.5- to 5.3-fold in the two NTHK1-transgenic lines in comparisonto their expression in Col-0 plants under normal con-dition. Under salt stress, whereas the AtERF4 and theCor6.6 were induced by approximately 2.5- to 3.3-foldin Col-0 plants, their expression was superinducedby approximately 5- and 11-fold, respectively, in thetransgenic lines compared with the nonstressed Col-0plants. Slightly higher expressions of the rd17 gene inthe two transgenic lines were also observed (Fig. 5A).For the DREB2A, Erd10, and P5CS genes, no significantFigure 4. Relative root growth of the NTHK1-transgenic plants and the

ethylene-response mutants under salt stress. A, Relative root length ofthe wild type and the NTHK1-transgenic lines (S1 and S10) upontreatment with different concentrations of NaCl. B, Relative root lengthof 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, barsindicate SD. The ratio of the average root length (from 20 seedlings) fromsalt plate to the average root length (from 20 seedlings) from MS platewas defined as relative root length. Each data point represents averageof three independent experiments.

Figure 5. Expression of salt-responsive genes in the NTHK1-transgenicplants 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 NaClfor 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 indicatedtimes. C, NTHK1-inhibited gene expressions. Seedlings were subjectedto treatment with water or 100 mM NaCl for 6 h and total RNA wasisolated. BBC1, Lea, and AtNAC2 genes, identified from a microarrayanalysis, were used as probes for RNA-blot analysis.

Cao et al.





difference in expression was observed between thetwo transgenic lines and the control plants, althoughthese genes showed slight inductions under salt-stresscondition, indicating that these genes were not regu-lated by the NTHK1 gene (Fig. 5A). It was interestingto find that the NTHK1 mRNA was accumulated undersalt stress (Fig. 5A), and this phenomenon will bestudied in the following text (Fig. 6).

The Arabidopsis ethylene-response mutants werealso investigated for their effects on expression of theNTHK1-activated genes (Fig. 5B). Under normal con-dition, 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 andein4-1, but not in ein2-1. For AtERF4, its expression wasin a similar level in all the four plants under normalcondition. Under salt-stress condition, Cor6.6 expres-sion was induced in all the plants tested, and theinduction level was the strongest in etr1-1. For rd17expression, the induction was stronger in etr1-1 andein4-1 than that in ein2-1. For AtERF4 expression, theinduction was earlier in etr1-1, ein4-1, and ein2-1 thanthat in Col-0 plants. These results indicate that gain-of-function mutation of the ethylene receptors ETR1 andEIN4 can enhance expression of Cor6.6, rd17, andAtERF4. EIN2 may be a negative regulator for Cor6.6and AtERF4.

NTHK1 also down-regulated gene expressions. Fig-ure 5C shows the expressions of three genes that wereidentified in a microarray analysis using salt-stressedCol-0 and salt-stressed NTHK1-transgenic plants (Heet al., 2005). BBC1 (At3g49010) encodes 60S ribosomalprotein L13 (RPL13B)/breast basic conserved protein1-related. This gene expression was lowered by saltstress and further reduced in the NTHK1-transgencicline S10. On the contrary, the Lea and the AtNAC2 gene(He et al., 2005) were salt-inducible. However, theinductions of the two genes were reduced in intensityin the NTHK1-transgenic plants when subjected to salttreatment (Fig. 5C).

NTHK1 mRNA Accumulation in the Transgenic Plantsunder Various Treatments

The NTHK1 mRNA in the transgenic plants wasaccumulated under salt stress (Fig. 5A). We furtherinvestigated the mRNA level of this gene in the trans-genic line S10 in response to salt and other treatments.It can be seen in Figure 6A that the NTHK1 mRNA waspresent in a relatively low level in the transgenic plants(S10). However, it was steadily accumulated upontreatment with increasing concentrations of NaCl. Inwild-type plants, no signal was detected. Time-coursestudy further demonstrated the induction of NTHK1by salt stress, and ACC exerted no effect on the NTHK1transcript level (Fig. 6B). The NTHK1-transgenic plantswere also treated with other stresses and plant hor-mones or chemicals, and Figure 6C shows that theNTHK1 mRNA levels were not affected by treatmentswith polyethylene glycol, low temperature (4�C), heatshock (37�C), or wounding. Abscisic acid showed noinfluence on NTHK1 gene expression either. However,treatment with cycloheximide (CHX), a protein syn-thesis inhibitor, resulted in dramatic accumulation ofthe NTHK1 transcripts (Fig. 6C), indicating that eitherde novo protein synthesis may not be required for theNTHK1 accumulation or a labile suppressor of NTHK1expression requires de novo protein biosynthesis. Thetransgenic plants were also subjected to treatmentswith other salts, and Figure 6C shows that the NTHK1accumulation was specifically induced by Na1 treat-ments but not significantly affected by K1, Li1, Cl2,and other anions tested.

DISCUSSION

Ethylene signaling is important in regulating plantgrowth and stress responses, and ethylene functionsthrough its receptors. Although it is generally believedthat ethylene signaling functions in multiple stressresponses, it is not clear what specific roles the recep-tors can play under salt stress. In this study, we trans-formed a tobacco type II ethylene receptor homologgene NTHK1 into Arabidopsis and found that theresulting transgenic plants, with NTHK1 mRNA andprotein expression, were salt sensitive as can be seen

Figure 6. NTHK1 gene expression in the NTHK1-transgenic plants inresponse to salt and other treatments. A, NTHK1 expression in the S10transgenic line after treatment with increasing concentrations of NaClfor 12 h. B, NTHK1 expression in the S10 transgenic line after treatmentwith 100 mM NaCl, or 100 mM NaCl plus 100 mM ACC. C, NTHK1expression in the S10 transgenic line upon treatment with variousabiotic stresses, chemicals, or different salts for 6 h. For all the sections,‘‘rRNA’’ indicates the 18S rRNA gene.






from the severe epinasty phenotype, high electrolyteleakage, and reduced root growth under salt stress.Epinasty phenotype has been reported to correlatewith the severity of salt stress ( Jones and El-Beltagy,1989; El-Iklil et al., 2000). Because overexpression ofNTHK1 in the transgenic plants seems to representgain of function of ethylene receptor, we speculate thatthe receptor function would lead to the salt-sensitiveresponses. The salt-sensitive epinasty phenotype andthe high electrolyte leakage can be fully or partiallysuppressed by ACC treatment, suggesting that ethyl-ene exerts a negative effect on ethylene receptors. Thisfact also suggests that ethylene may be required forplant salt tolerance. The gene expression ratios be-tween the salt-stressed transgenic and salt-stressedCol-0 plants, identified by microarray analysis, canalso be reversed by ACC to the normal ratios underMS condition (data not shown; Cao, 2004). This effectconferred by ACC application further confirmed thenegative regulatory mechanism between ethylene andits receptors (Hua and Meyerowitz, 1998). In addition,ACC can rescue the salt-stressed phenotype in wild-type plants, further implying that ethylene signaling isbeneficial for plant survival under salt stress.

The ethylene receptor gain-of-function mutants etr1-1and ein4-1 exhibited salt sensitivity as observed fromtheir severe epinasty phenotype, the high leakagelevels, and reduced root growth, indicating that re-ceptor functions may result in sensitive responsesupon NaCl treatment. However, loss-of-function mu-tants ein4-7, etr1-6, and etr1-8 of these receptors do nothave apparent improvement in salt tolerance in com-parison with the wild-type plants, possibly implyingthat loss of a single receptor does not significantlyaffect the plant responses to salt stress. This fact may beconsistent with the report that single loss-of-functionreceptor mutants did not have ethylene-response phe-notype (Hua and Meyerowitz, 1998). However, anETR1 single loss-of-function mutant etr1-7 has beenfound to have enhanced sensitivity and exaggerated re-sponse to ethylene (Cancel and Larsen, 2002). Whetherthis mutant has any tolerance to salt stress remains tobe investigated.

The ethylene-signaling mutant ein2-1 also exhib-ited sensitivity under salt stress, indicating that EIN2promotes plant salt tolerance. EIN2 is a membrane-associated protein and plays central roles in the ethylene-signaling pathway (Alonso et al., 1999). This proteinis essential for the salt-induced expression of theAtNAC2 gene, which promotes lateral root formation(He et al., 2005). Another ethylene insensitive mutant,ein3-1, did not have the phenotypic change under saltstress in comparison with the wild-type plants, sug-gesting that EIN3, a transcription factor in the ethylene-signaling pathway (Chao et al., 1997), is not requiredfor the plant phenotypic response to salt stress. Thesignaling pathway may have switched to a branchpathway before EIN3 and then led to the salt-inducedphenotypic responses. Alternatively, other EIN3 ho-mologs may mediate these salt-induced phenotypic

changes. It should be noted that although EIN3 ap-pears not to be involved in phenotypic changes undersalt stress, it may still play some roles for salt tolerancein the physiological aspects as can be seen from thehigh electrolyte leakage in the salt-stressed ein3-1 mu-tant. Slightly lower survival rate has been observed inthe ein3-1 mutant under high salinity stress whencompared with the wild-type control, suggesting a rolefor EIN3 in salt tolerance (Achard et al., 2006). Muta-tion of the EIN3-degradation pathway componentsEBF1 and EBF2 (Guo and Ecker, 2003; Potuschak et al.,2003) also results in salt tolerance, and EIN3 maypromote salt tolerance by enhancing DELLA function(Achard et al., 2006). In this study, we did not find asignificant change in salt response in the ctr1-1 mutanttreated with 100 mM NaCl, although salt toleranceis expected in this mutant according to its constitu-tive ethylene-response trait. However, under higher(200 mM) salinity, the ctr1-1 mutants exhibited highersurvival rate in comparison with the wild-type plants(Achard et al., 2006), consistent with the speculationthat ethylene signaling is important for plant salttolerance. It is possible that severe salt stress coulddetect the survival difference between wild type andthe ctr1-1 mutant. Although ctr1-1 is tolerant to highsalt, the present ACC-treated wild type can only tol-erate to 150 mM NaCl. The reason for the difference isnot known. It is possible that the wild type needs tomanage the whole situation to make a balanced deci-sion for later generation, whereas the mutant may beonly survived. This phenomenon remains to be furtherinvestigated.

Overexpression of the NTHK1 gene results in longseedlings and large rosettes in transgenic plants incomparison with the wild type. Question may arise asto whether these phenotypes were caused by ethyleneinsensitivity or were a consequence of cell enlarge-ment caused by NTHK1. It is apparent that the etr1-1mutant seedling length is more insensitive to ethylenethan the NTHK1-transgenic plants (Fig. 1E; Zhou et al.,2006), whereas their rosette sizes are comparable. Inthe presence of ethylene biosynthesis blocker AVG, thetransgenic seedlings showed similar length to the Col-0seedlings, possibly suggesting that NTHK1 could re-sult in reduced ethylene sensitivity. It is possible thatNTHK1 could lead to the reduced ethylene sensitivityin seedling length but cause cell enlargement in rosetteleaves, and the rosette size is not related to ethylenesensitivity. The ethylene emission was also measuredin seedlings of the transgenic plants and the wild-typeplants, and no significant difference was detected(data not shown), further implying that the longerseedling of the transgenic plants is due to the reducedethylene sensitivity.

Although the NTHK1-transgenic plants showedreduced sensitivity to ethylene, they were still respon-sive to ethylene because the NTHK1 still had the nor-mal ethylene-binding site. Therefore, ACC can rescuethe salt-stressed phenotype through ethylene bindingto the receptors. On the contrary, the etr1-1 mutant

Cao et al.





was completely insensitive to ethylene because of themutation in the ethylene-binding site of ETR1 andethylene cannot bind to the mutated ETR1. Therefore,ACC cannot rescue the salt-stressed etr1-1. The presentresults that overexpression of the ethylene receptorgene NTHK1 led to reduced ethylene sensitivity maybe consistent with previous reports. Ciardi et al. (2000)found that tomato ethylene receptor NR overexpres-sion reduced ethylene sensitivity in seedlings andmature plants. Consistently, transgenic plants withreduced LeETR4 gene expression displayed multiplesymptoms of extreme ethylene sensitivity (Tiemanet al., 2000).

Salt injury to plants can be estimated by the relativeelectrolyte leakage, and more injury would lead tohigher level of relative electrolyte leakage. Electrolyteleakage allows assessment of the intactness of cellmembranes, and more leakage indicates more damageof the membrane system (Verslues et al., 2006). TheNTHK1-transgenic plants and the ethylene-responsemutants etr1-1, ein4-1, and ein2-1 all had higher levelsof the relative electrolyte leakage under salt stress,consistent with the salt sensitivity observed from thesalt-induced epinasty and inhibited root growth. ACCcan partially suppress the salt-caused electrolyte leak-age in NTHK1-transgenic plants, indicating that eth-ylene is required for plant tolerance response to saltstress. It should be noted that the ein2-1 mutant hadthe highest level of relative electrolyte leakage undersalt stress when compared with other mutants, sug-gesting that EIN2 is the most important componentin signaling salt-tolerant responses. It should also benoted that the ein3-1 mutant did not exhibit salt-induced epinasty or inhibited root growth under saltstress but still had high level of electrolyte leakage.This fact suggests that the phenotypic change wasseparated from the electrolyte leakage. It is possiblethat the ethylene receptor and EIN2 can regulate bothaspects, whereas EIN3, a downstream factor, can onlycontrol electrolyte leakage but not phenotypic change.

NTHK1 enhances expression of salt-responsive genesAtERF4, Cor6.6, and rd17, indicating its role in salt-stress response. Constitutive receptor signaling inArabidopsis ethylene receptor gain-of-function mu-tants etr1-1 and ein4-1 also promotes expression ofthese genes. Recently, AtERF4 has been found to be atranscriptional repressor conferring ethylene insensi-tivity in its transgenic Arabidopsis plants, and theAtERF4-overexpressing plants are hypersensitive tosodium chloride (McGrath et al., 2005; Yang et al.,2005). This observation is consistent with our presentfindings. Therefore, gain of function of ethylene recep-tors may confer ethylene insensitivity and salt sensi-tivity at least partially through activation of the AtERF4gene. In addition to the activation of some gene ex-pressions, NTHK1 also down-regulates expressions ofsalt-induced or salt-inhibited genes (Fig. 5C). Previ-ously, we have found that a NAC-type transcriptionfactor gene, AtNAC2, was salt inducible and involvedin lateral root development (He et al., 2005). The salt

induction of the AtNAC2 gene was reduced in intensityin 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 reg-ulate salt-stress response by controlling multiple geneexpressions.

The NTHK1 gene is salt inducible in tobacco plants(Zhang et al., 2001a). The NTHK1 transcripts were alsoaccumulated in the NTHK1-transgenic lines under salt-stress condition. Recently, we have identified that thesalt-responsive element was likely located in the trans-membrane-coding region of the NTHK1 gene (Zhouet al., 2006). It is possible that salt stress induces a com-ponent that can stabilize the NTHK1 mRNA by bindingto the specific sequence. Alternatively, salt stress mayinhibit a transcription repressor that usually binds tothe salt-responsive element under normal condition,resulting in the transcript accumulation under saltstress. Another possibility is that salt stress inhibitedthe mRNA-degradation enzymes. Treatment with pro-tein synthesis inhibitor CHX resulted in an even drasticaccumulation of NTHK1 mRNA, indicating that eitherNTHK1 transcript accumulation does not require denovo protein biosynthesis or a labile suppressor ofNTHK1 expression requires de novo protein biosyn-thesis. Because the expression of NTHK1 is driven bythe constitutive cauliflower mosaic virus 35S promoter,the NTHK1 transcript is more likely regulated post-transcriptionally by the latter mechanism. In animalcell systems, degradation of mature mRNA is a regu-lated process that determines gene expression. cis-Regulatory elements have been found in the 5#-UTR,the coding region, and the 3#-UTR. trans-Acting pro-teins have also been identified and can affect the mRNAstability by interacting with the cis-elements (Holcikand Liebhaber, 1997). In plants, the cis-element respon-sible for the CHX-induced SAUR mRNA accumulationwas localized in the open reading frame of this gene (Liet al., 1994). A Na1/H1 antiporter SOS1 gene, driven bythe 35S promoter, has also been observed to haveincreased transcript level in transgenic Arabidopsisupon NaCl treatment (Shi et al., 2003).

Salt-induced accumulation of the NTHK1 transcriptshas also been observed in transgenic tobacco plantsoverexpressing NTHK1 (Cao et al., 2006), indicating aconsistency with the case in the present transgenicArabidopsis. It is not known why a gene that leads to asalt-sensitive phenotype is induced upon salt treat-ment. It is possible that salt stress-caused inhibition orslower growth of a plant is an adaptive feature thatwould make plants grow slowly under stress and thussave resources for later recovery after the stress hasbeen removed or alleviated, as suggested by Zhu(2001). In the present case, the NTHK1 may first sensethe salt stress and slow down the growth. At the sametime, the NTHK1 can also activate expression of salt-responsive genes that are beneficial for plant survivalunder salt stress. Later, the accumulation of ethylenewill inhibit the NTHK1 action and gradually recoverthe growth.






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

Ethylene has been regarded as a stress hormoneand is induced by many stresses (Abeles et al., 1992;Morgan and Drew, 1997). However, its role in saltstress is equivocal. El-Iklil et al. (2000) has reportedthat lower ethylene production was associated withsalt tolerance. On the contrary, higher ethylene pro-duction has been regarded as an indicator for salttolerance in rice (Khan et al., 1987). Ethylene has longbeen recognized as a growth inhibitor, but it can alsopromote growth (Pierik et al., 2006). A recent studysuggests that ethylene signaling promotes salt toler-ance in Arabidopsis (Achard et al., 2006). This studysuggests that ethylene receptor function leads to saltsensitivity and ACC appears to suppress this saltsensitivity, implying that ethylene signaling is re-quired for salt tolerance. Plant response to salt stressmay depend on the balance and/or interaction be-tween receptor and ethylene. When receptor signalingis prevalent, the plant is sensitive to salt stress butshows large rosette and late flowering. When ethylenesignaling is prevalent, the plant is tolerant to salt stressbut has small rosette and early flowering. The plantneeds to make adjustment between these two extremesituations. Fine-tuning at various levels may makeplants in an active homeostasis, and then the plantscan survive better under stress condition and haverelatively 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 3 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 V 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 infiltra-

tion method. The transgenic plants were screened on MS agar medium

containing 50 mg/L Kanamycin. The presence and integration of the trans-

gene 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 com-

pared 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 mM or 100 mM) 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 mM 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 mM abscisic acid originally dissolved in dimethyl sulfoxide (DMSO), 50 mM

CHX originally dissolved in DMSO, DMSO (100 mL/50 mL solution), and

100 mM 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 concen-

trations of ACC or 2 mM 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 mg) 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 a-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.

Cao et al.





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#-ATATGAG-

CACTTGGATCC-3#; for AtERF4, 5#-CTATCCGAGAATGGCCAAG-3# and

5#-AACAACATGGGGTGAAACC-3#; for Cor6.6, 5#-ACATCAAAAAC-

GATTTTACAAG-3# and 5#-GAACTTAAACTAGATTTTGTTG-3#; for Erd10,

5#-AGTTTCTCTTTATCATTCACG-3# and 5#-AATAAAAGAGACAATGA-

TCAAC-3#; for rd17, 5#-CTTAAAGCAACTACACAAGTC-3# and 5#-ATC-

ACAAAACACAGCGAATG-3#; and for P5CS, 5#-GACTAAGTTGACTC-

GTTCTC-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% glutar-

aldehyde 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 pheno-

type were further transferred onto 100 mM NaCl plus ACC (100 mM) 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 deter-

mined. 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.

ACKNOWLEDGMENTS

We thank Dr. Jian-Kang Zhu (University of California, Riverside) for

helpful comments on the manuscript. Thanks are also due to Dr. E.M.

Meyerowitz (California Institute of Technology), Dr. A.B. Bleecker (University

of Wisconsin, Madison), and Arabidopsis Biological Resource Center for

providing seeds of Arabidopsis ethylene-response mutants.

Received December 6, 2006; accepted December 11, 2006; published December

22, 2006.

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Modulation of Ethylene Responses Affects Plant Salt-Stress Responses