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First published online July 9, 2004; 10.1104/pp.104.039909 Plant Physiology 135:1697-1709 (2004) © 2004 American Society of Plant Biologists Comparative Genomics in Salt Tolerance between Arabidopsis and Arabidopsis-Related Halophyte Salt Cress Using Arabidopsis Microarray1Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, Tsukuba, Ibaraki 305–0074, Japan (T.T., M.S., Y.N., K.S.); Plant Mutation Exploration Team, Plant Functional Genomics Research Group, RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama 230–0045, Japan (M.S., M.S., T.S., M.N.); Genomic Knowledge Base Research Team, Bioinformatics Group, RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama 230–0045, Japan (M.S., T.S.); Experimental Plant Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305–0074, Japan (M.K., K.I.); Department of Biology, Tokyo Gakugei University, Koganei-shi, Tokyo 184–8501, Japan (Y.N., M.N.); and Institute of Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521 (J.K.Z.)
Salt cress (Thellungiella halophila), a halophyte, is a genetic model system with a small plant size, short life cycle, copious seed production, small genome size, and an efficient transformation. Its genes have a high sequence identity (90%–95% at cDNA level) to genes of its close relative, Arabidopsis. These qualities are advantageous not only in genetics but also in genomics, such as gene expression profiling using Arabidopsis cDNA microarrays. Although salt cress plants are salt tolerant and can grow in 500 mM NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation. This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes. To elucidate the differences in the regulation of salt tolerance between salt cress and Arabidopsis, we analyzed the gene expression profiles in salt cress by using a full-length Arabidopsis cDNA microarray. In salt cress, only a few genes were induced by 250 mM NaCl stress in contrast to Arabidopsis. Notably a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein,  -glucosidase, and SOS1, were expressed in salt cress at high levels even in the absence of stress. Under normal growing conditions, salt cress accumulated Pro at much higher levels than did Arabidopsis, and this corresponded to a higher expression of AtP5CS in salt cress, a key enzyme of Pro biosynthesis. Furthermore, salt cress was more tolerant to oxidative stress than Arabidopsis. Stress tolerance of salt cress may be due to constitutive overexpression of many genes that function in stress tolerance and that are stress inducible in Arabidopsis.
Abiotic stresses greatly affect plant growth and crop production. To survive against these stresses, plants respond and adapt with complex mechanisms, including developmental, morphological, physiological, and biochemical strategies. High salinity is a major abiotic stress causing both osmotic and ionic stress. The adaptive strategy to osmotic stress is the accumulation of osmoprotectants such as Pro, glycinebetaine, mannitol, and the raffinose family oligosaccharides. This strategy has been shown to be important in improving stress tolerance in plants by manipulating genes encoding key enzymes of the osmoprotectant synthesis or degradation pathway (Tarczynski et al., 1993  ; Kavi-Kishor et al., 1995; Thomas et al., 1995; Nanjo et al., 1999a, 1999b; Taji et al., 2002). A high level of Na+ is toxic to plants because it disturbs cytoplasmic K+/Na+ homeostasis. To prevent Na+ accumulation in the cytoplasm, plants use the following three strategies: reducing Na+ influx, vascuolar compartmentation of Na+, and excretion of Na+ via plasma membrane Na+/H+ antiporters (Ward et al., 2003). Overexpression of the vacuolar Na+/H+ antiporter NHX1 has been shown to confer salt tolerance in Arabidopsis and tomato, suggesting the utility of this vascuolar compartmentation of Na+ (Apse et al., 1999; Zhang and Blumwald, 2001). Also, overexpression of a plasma membrane Na+/H+ antiporter gene in a freshwater cyanobacterium conferred extreme salt tolerance and allowed growth in seawater, indicating the effectiveness of excreting Na+ via plasma membrane Na+/H+ antiporters for salt tolerance (Waditee et al., 2002). Furthermore, very recently, overexpression of SOS1, a plasma membrane Na+/H+ antiporter, was shown to confer salt tolerance by retrieving Na+ from the xylem of transgenic plants (Shi et al., 2003).
Although there have been many reports on salt tolerance, most of the studies have been on a typical glycophyte, Arabidopsis, and not on halophytes. This is because the technological advantages of this model plant are too significant, and halophytic plants have not been shown to be a suitable genetic and genomic model system. Recently, the halophytic plant species salt cress (Thellungiella halophila) was reported to grow in high salinity coastal areas in eastern China. Salt cress is thought to be a good model plant for the analysis of high salinity tolerance because it is closely related to Arabidopsis and has good genetic features such as similar morphology, small size, short life cycle, high seed number, and the ability to self-pollinate. Furthermore, salt cress has a gene composition with >90% nucleotide identity with that of Arabidopsis, and it can be transformed in planta according to the Arabidopsis protocol (Bressan et al., 2001
Although Arabidopsis is a typical glycophyte that is not particularly salt tolerant, a number of recent studies suggest that it may contain most, if not all, of the salt tolerance genes that one might find in halophytes (Shinozaki and Yamaguchi-Shinozaki, 2000 In this study, we applied the full-length Arabidopsis cDNA microarray to reveal the differences in the regulation of salt tolerance mechanisms between a glycophyte, Arabidopsis, and a halophyte, salt cress. Furthermore, we compared the expression profiling data of salt cress with our previous Arabidopsis expression profiling data of genes that included not only those obtained by various abiotic stress treatments but also those obtained by biotic stress-related treatments using the same full-length Arabidopsis cDNA microarray. Salt stress tolerance of salt cress is discussed by comparison of Arabidopsis based on gene expression profiling of stress-inducible genes.
Salt Cress Is Extremely Salt Tolerant The germination rate of salt cress in the absence of 4°C stratification was very low and not uniform. Stratification treatment at 4°C for 1 week greatly improved the low germination rate. Growth of the salt cress seedling was slower than that of Arabidopsis. Thus, it was necessary to examine the salt stress tolerance of salt cress and Arabidopsis either with seedlings of the same size or at the same age. Three-week-old Arabidopsis and 4-week-old salt cress plants were exposed to 500 mM NaCl solution to investigate the salt tolerance with seedlings of the same size. The salt tolerance of seedlings at the same age was examined using 3-week-old Arabidopsis and the salt cress plants exposed to 500 mM NaCl solution. After 3 weeks of treatment, complete chlorosis was observed in all the Arabidopsis plants of the same size and same age. By comparison, the salt cress plants were not affected by either condition (Fig. 1, A and B ).
Salt Tolerance in the Hydroponic Culture System and Differences in NaCl Uptake
The demonstration of extreme salt tolerance of salt cress plants implies the existence of important mechanisms for salt tolerance in the root. It was necessary to gather not only the rosette leaves but also the roots. However, we could not harvest the root of the soil-grown Arabidopsis and salt cress plants. So we adopted the hydroponic culture system with glass beads (Nanjo et al., 1999a
To investigate the importance of salt cress roots in the tolerance to salt stress, we measured the accumulation of NaCl in the leaves of Arabidopsis and salt cress plants during high salinity stress in the hydroponic culture system. Three-week-old Arabidopsis and 4-week-old salt cress plants were exposed to 250 mM NaCl solution for 2, 5, 10, and 24 h. After exposure for 10 or 24 h, NaCl uptake of salt cress plants became slower than that of Arabidopsis (Fig. 2B). This suggested that salt cress roots possess special mechanisms to prevent inflow of NaCl.
To analyze the molecular mechanisms of salt tolerance in salt cress, we compared gene expression profiles in salt cress with those of Arabidopsis by using a full-length cDNA microarray (approximately 7,000 Arabidopsis genes). The Arabidopsis full-length cDNA microarray is expected to hybridize mRNA of salt cress more efficiently than Arabidopsis oligonucleotide microarray because the oligonucleotide microarray needs strict identity between mRNAs in comparison with the full-length cDNA microarray. In addition, since salt cress genes share >90% nucleotide identity with Arabidopsis genes, efficient cross-hybridization was expected. To identify salt stress-inducible genes in salt cress and Arabidopsis, we treated plants with salt at 250 mM NaCl for 2 h. The reasons for using this condition are as follows: (1) genes rapidly inducible should be important for salt tolerance; (2) significant chlorosis is not observed within 2 h in control Arabidopsis; and (3) NaCl is taken up more efficiently at 10 h of salt treatment in Arabidopsis. The salt stress-inducible genes in salt cress and Arabidopsis were identified by microarray analyses that hybridized with Cy3 and Cy5 fluorescence-labeled probe pairs of salt-stressed plants plus unstressed plants. Subsequently, we investigated the differences in salt-inducible genes between salt cress and Arabidopsis by comparing their microarray data. Down-regulated genes could not be evaluated precisely because of lower hybridization signals. These genes might include not only down-regulated genes but also ortholog genes with low homology. Therefore, we concluded that the expression data obtained with heterologous microarray analysis can be used for the identification of up-regulation of genes.
Tables I and II show the genes that are up-regulated (ratio = log2 Cy5/Cy3
Genes Up-Regulated in Salt Cress Compared with Arabidopsis under Normal Growth Conditions
To compare expression profiles under normal growth conditions between salt cress and Arabidopsis, we hybridized the full-length Arabidopsis cDNA microarrays with Cy3 and Cy5 fluorescence-labeled probe pairs of nonstressed salt cress plus nonstressed Arabidopsis plants. Interestingly, a number of abiotic or biotic stress-inducible genes were expressed under normal growth conditions in salt cress (Table III; Fig. 4). Especially, the tendency observed in the genes up-regulated to high levels, and five of the eight genes with log2 ratios greater than 2.5 are known to be important genes in abiotic stress or biotic stress tolerance. The up-regulated genes are Fe-superoxide dismutase (SOD); 9-cis-epoxycarotenoid dioxygenase (AtNCED2); chitinase; plant defensin1.2 (PDF1.2);
We have performed many kinds of microarray analyses to analyze gene expression profiles in response to various abiotic, biotic stress, and oxidative stress in Arabidopsis using this full-length Arabidopsis cDNA microarray (Seki et al., 2002a , 2002b; Narusaka et al., 2003). Using the database of those microarray analyses, we analyzed the expression of the genes in Table III that were highly expressed (ratio;  1.5) in salt cress under normal growth conditions with Arabidopsis genes induced by various abiotic, biotic, and oxidative stress treatments. The abiotic stresses include drought, salt (NaCl), cold, and abscisic acid (ABA) treatment. The biotic and oxidative stress treatments include inoculation with a fungal pathogen, Alternaria brassicicola, to wild-type Arabidopsis (Columbia) and pad3-1 mutant (Glazebrook and Ausubel, 1994), signal molecules (salicylic acid [SA], jasmonic acid [JA], and ethylene [ET]), reactive oxygen species-inducing compounds (paraquat and rose Bengal), and UV-C treatment, which causes cell death (Narusaka et al., 2003). Figure 4 shows the results of cluster analysis of the resultant expression profiles. The expression profiles include five groups, i.e. abiotic stress-inducible genes, biotic stress-inducible genes, both abiotic and biotic inducible genes, abiotic and biotic noninducible genes, and abiotic and biotic suppressible genes (Table III). Interestingly, one-half of the genes belonged to the groups that were abiotic, biotic, or both abiotic and biotic stress-inducible genes in Arabidopsis (Fig. 4, A and B). The fact that many stress-inducible genes were expressed under normal growth conditions in salt cress might account for the relatively fewer salt stress-inducible genes compared to those of Arabidopsis with similar ratios.
We performed RNA gel-blot analysis on expression profiles of SOS1 and PDF1.2 genes to confirm the validity of the microarray analyses (Fig. 5). The expression levels of SOS1 and PDF1.2 in salt cress were higher than those of Arabidopsis by RNA gel-blot analysis as well as the microarray analysis. Especially, the expression level of PDF1.2 was very high in salt cress under a normal growth condition or salinity stress, whereas that in Arabidopsis was not detectable under these conditions. Therefore, the expression data obtained by microarray analysis were in good agreement with those obtained by RNA gel-blot analysis. When we performed RNA gel-blot analysis of other genes, such as P5CS using ThP5CS cDNA (AtP5CS ortholog in salt cress [accession no. BM985832]), the expression of P5CS was higher in salt cress. However, when we used Arabidopsis P5CS cDNA as a probe, we could not detect higher expression of P5CS in salt cress (data not shown). The disagreement between the microarray analysis and the RNA gel-blot analysis may have arisen because of the difference in their hybridization efficiency and gene family.
ABA and Pro Contents in Salt Cress AtNCED2 and AtP5CS genes were expressed at higher levels in nonstressed salt cress. NCED and P5CS encode key enzymes of ABA and Pro biosynthesis, respectively. Thus, we measured the accumulation of endogenous levels of ABA and Pro in salt cress and Arabidopsis under normal growth conditions in a hydroponic culture. The ABA content of salt cress was slightly higher than that of Arabidopsis (Fig. 6A). The Pro content in salt cress was markedly higher than that of Arabidopsis during normal growth conditions in a hydroponic culture. Therefore, the extreme stress tolerance to high salinity characteristic of salt cress is due in part to the overaccumulation of Pro under unstressed conditions.
Salt Cress Is Tolerant to Oxidative Stress
The SOD gene was expressed at much higher levels in nonstressed salt cress, suggesting that salt cress is tolerant to oxidative stress. SOD is one of the key enzymes in the protective system against oxidative stress since it catalyzes the dismutation of the superoxide radical to O2 and H2O2, a reaction that constitutes the first cellular defense against many oxidative stress situations. To analyze the oxidative tolerance in salt cress, we examined the paraquat tolerance of salt cress and Arabidopsis. Paraquat is known to cause the formation of a superoxide anion and a singlet oxygen (Lin and Culotta, 1995
It is now hypothesized that the mechanisms of salt tolerance in halophytes are substantially the same as those known to exist in glycophytes and that subtle differences in regulation result in large variations in tolerance or sensitivity (Zhu, 2001 ). In this study, we investigated the strategy used for salt tolerance in a halophyte, salt cress, which is a close relative of Arabidopsis, by using the genomic information of Arabidopsis. Comparison of EST sequences from Arabidopsis and salt cress revealed a high DNA sequence identity (90%–95%) for the majority of transcripts, which indicates that the full-length cDNA microarray of Arabidopsis can be used for expression profiling of salt cress genes. As a result of microarray analyses, only 6 genes were shown to be strongly induced in response to high salinity stress in salt cress, whereas 40 genes were identified as salt stress-inducible genes in Arabidopsis (Tables I and II; Fig. 3). Many abiotic or biotic stress-inducible genes with various functions were expressed under normal growth conditions in salt cress than in Arabidopsis. This may be due to only six inducible genes detected in salt cress. Especially, the tendency observed in the genes up-regulated to high levels, and five of the eight genes with log2 ratios greater than 2.5 were known to be important genes in abiotic stress or biotic stress tolerance. As a result of hierarchical clustering among the genes highly expressed in salt cress without stress and the stress-inducible genes in Arabidopsis under different stress conditions, it was revealed that one-half of the genes highly expressed in salt cress belong to groups of genes that were abiotic, biotic, or abiotic and biotic stress-inducible in Arabidopsis (Fig. 4A). Furthermore, the expression profiles of genes highly expressed in salt cress under normal growth conditions resembled those of Arabidopsis genes induced under abiotic stresses rather than biotic stresses (Fig. 4B). However, some important genes involved in biotic stress tolerance were also observed in genes highly expressed in salt cress without stress. These observations suggest that salt cress is tolerant to biotic stresses as well as abiotic stresses because of constitutive overexpression of stress-related genes under unstressed conditions. These results indicate that salt cress does not immediately respond to a variety of environmental stresses, including high salinity at the transcriptional level, but instead adapted to them by expressing a number of genes that are known components of abiotic or biotic stress defense and survival machinery even in the absence of these stresses (Table III; Fig. 4). The genes overexpressed in the salt cress under normal conditions include Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, -glucosidase, and SOS1 (see Table III). Possible functions of their gene products in stress tolerance of salt cress are discussed in the following.
Pro, the most common osmoprotectant, accumulates in many organisms, including higher plants exposed to environmental stresses such as high salinity, drought, and freezing. Some reports have even indicated a positive correlation between Pro accumulation and the acquisition of stress tolerance (Kavi Kishor et al., 1995
The Fe-SOD gene was overexpressed in salt cress under normal conditions, and salt cress showed a higher tolerance to paraquat treatment (Fig. 6). SODs are essential components in almost all plant antioxidant defense mechanisms. The SOD isoenzymes can be classified into Cu/Zn, Mn, and Fe types according to their metal cofactor. Plants generally contain Fe-SOD and/or Cu/Zn-SOD in the chloroplasts (Holmberg and Bulow, 1998
With the acquisition of salt tolerance, there is no subsequent Na+ accumulation in the cytoplasm. Theoretically, there are three mechanisms: (1) reduction of Na+ influx; (2) vascuolar compartmentalization of Na+; and (3) excretion of Na+ via plasma membrane Na+/H+ antiporters. Overexpression of the vacuolar Na+/H+ antiporter NHX1 has been shown to confer salt tolerance in Arabidopsis and tomato, suggesting the utility of this vascuolar compartmentation of Na+ (Apse et al., 1999
Interestingly, in salt cress, not only abiotic but also biotic stress-inducible genes were overexpressed under normal growth conditions (Table III). Pathogenesis-related protein PDF1.2, used as a general marker gene for pathogenesis, chitinase, P-protein, and In conclusion, we showed that salt cress had not only salt stress tolerance but also oxidative stress tolerance. Based on expression profiling using the full-length Arabidopsis cDNA microarray, we showed that various genes induced by abiotic and biotic stresses in Arabidopsis are overexpressed in unstressed conditions in salt cress. This suggests that stress-inducible signaling pathways are constitutive and active in salt cress even under normal growth conditions without stress.
Plant Material and Salt Stress Treatments for Soil-Grown Plants Salt cress (Thellungiella halophila) and Arabidopsis L. Heynh., ecotype Columbia, were sown on MS plates containing 1.2% (w/v) agar and 3% Suc. The seeds were stratified at 4°C for 7 d and then transferred to 22°C under continuous light for germination and growth. Two or 3 weeks after germination, seedlings of Arabidopsis and salt cress were transferred onto separate 9-cm plastic pots filled with a 1:1 perlite:vermiculite and watered with 1,000-fold diluted Hyponex (Hyponex, Osaka). One week after transfer, the seedlings were transferred into a 1,000-fold diluted Hyponex solution containing 500 mM NaCl for the salinity stress treatment.
In addition to the above method, two- or 3-week-old seedlings of Arabidopsis and salt cress were transferred onto tea strainers filled with glass beads with a hydroponic culture (1,000-fold diluted Hyponex). One week after transfer, the tea strainers containing the seedlings were transferred into 1,000-fold diluted Hyponex containing 250 mM NaCl for salinity stress.
Three-week-old Arabidopsis and 4-week-old salt cress plants grown in the hydroponic system were exposed to 250 mM NaCl solution for 2, 5, 10, and 24 h. We sampled only rosette leaves because this portion was immersed in the hydroponic culture. Na+ content was determined by converting the result of electrolyte leakage from leaves into the Na+. A total of 0.1 g leaves from each treatment group were immersed in 5 mL of sterile distilled water. The solution was measured for conductivity with a conductivity meter (DS-15; Horiba, Kyoto) after the sample was boiled for 14 min.
Three-week-old Arabidopsis and 4-week-old salt cress plants grown in the hydroponic system were ground in distilled water. The homogenate was boiled for 6 min and then centrifuged at 10,000g for 15 min at 4°C. The supernatant was precipitated with 10% TCA for 5 h and centrifuged at 10,000g for 20 min. The supernatant after TCA precipitation was derivatized, and the contents of free Pro were determined by HPLC (model LC Module I plus; Waters, Milford, MA).
Three-week-old Arabidopsis and 4-week-old salt cress plants grown in the hydroponic system were used as samples. Endogenous ABA was measured using exactly the same procedure as previously described (Iuchi et al., 2000
Total RNA was prepared using TRIZOL reagent (Life Technologies, Rockville, MD), and mRNA was prepared using an mRNA isolation kit (Miltenyi Biotec, Auburn, CA). Isolated total RNA was also used for RNA gel-blot analysis. Total RNA was fractionated in a 1% agarose gel containing formaldehyde and was blotted onto a nylon filter. The filters were hybridized with 32P-labeled fragments at 57°C, washed twice with 0.1x SSC, 0.1% SDS, at 60°C for 15 min, and autoradiographed.
Full-length cDNA microarray analysis was carried as reported previously (Seki et al., 2001
Image analysis and signal quantification were performed with QuantArray version 2.0 (GSI Lumonics, Oxnard, CA). Background fluorescence was calculated on the basis of the fluorescence signal of the negative control genes, the mouse nicotinic acetylcholine receptor epsilon-subunit (nAChRE) gene and the mouse glucocorticoid receptor homolog gene. Intensity-dependent normalization (Yang et al., 2002
We thank Dr. Tokihiko Nanjo for discussion, Setsuko Kawamura for her excellent technical assistance, and Emily Smith-Katai for critical reading of the manuscript. Received January 29, 2004; returned for revision April 7, 2004; accepted April 8, 2004.
1 This work was supported in part by the Genome Research Project in RIKEN (to K.S.) and by the Special Postdoctoral Researchers' Program from RIKEN (to T.T.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.039909. * Corresponding author; e-mail sinozaki{at}rtc.riken.go.jp; fax 81–29–836–4359.
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ABSTRACT
RESULTS
-1-pyrroline-5-carboxylate synthetase (AtP5CS); plasma membrane Na+/H+ antiporter (SOS1); P-protein associated with nitric oxide (NO) production; and 
-galactosidase enhances freezing tolerance in transgenic petunia. Plant Physiol 133: 901–909
