JXB Advance Access originally published online on September 10, 2004
Journal of Experimental Botany 2004 55(406):2213-2218; doi:10.1093/jxb/erh242
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RESEARCH PAPER |
Osmotic stress in barley regulates expression of a different set of genes than salt stress does
1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
2Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
3State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, People's Republic of China
* To whom correspondence should be addressed. Fax: +63 2 845 0606. E-mail: J.BENNETT{at}CGIAR.ORG
Received 2 February 2004; Accepted 7 July 2004
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Abstract |
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Under high salt conditions, plant growth is severely inhibited due to both osmotic and ionic stresses. In an effort to dissect genes and pathways that respond to changes in osmotic potential under salt stress, the expression patterns were compared of 460 non-redundant salt-responsive genes in barley during the initial phase under osmotic versus salt stress using cDNA microarrays with northern blot and real-time RT-PCR analyses. Out of 52 genes that were differentially expressed under osmotic stress, 11, such as the up-regulated genes for pyrroline-5-carboxylate synthetase, betaine aldehyde dehydrogenase 2, plasma membrane protein 3, and the down-regulated genes for water channel 2, heat shock protein 70, and phospholipase C, were regulated in a virtually identical manner under salt stress. These genes were involved in a wide range of metabolic and signalling pathways suggesting that, during the initial phase under salt stress, several of the cellular responses are mediated by changes in osmotic potential.
Key words: Barley, cDNA microarray, gene expression, osmotic stress, salt stress
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Water is one of the most essential elements for all living organisms. In plants, transpiration of water is useful in preventing temperature increases. In grass plants in particular, more than 90% of water uptake from the soil is consumed by transpiration during the hot summer season. Therefore, plant growth is severely inhibited under water stress conditions (Yeo et al., 1991
; Pérez-Alfocea et al., 1993). Water stress, caused by drought and/or soil salinity, triggers decreasing cell turgor pressure, and then wilting. To enable water uptake from the soil under water stress conditions, plants synthesize various kinds of osmoprotectants, such as glycinebetaine and proline (Gorham et al., 1985; Delauney and Verma, 1993). It is considered that the accumulation of osmoprotectants leads to improving osmotic stress tolerance (Kishor et al., 1995). The effectiveness of osmotic adjustment by osmoprotectants was proved using transgenic plants, which accumulated higher concentrations of proline, glycinebetaine, or pinitol (Kishor et al., 1995; Sheveleva et al., 1997; Takabe et al., 1998). On the other hand, it was reported that the regulation of water permeability through water channels also modified the sensitivity to water stress (Aharon et al., 2003). Thus, osmotic adjustments by both the accumulation of osmoprotectants and the regulation of water permeability play an important role for tolerance toward water stress.
cDNA microarray has been developed as a tool for comprehensive expression analysis, providing information on gene expression profiling. Many papers using microarray technology have described changes in the transcriptome of model plants, especially Arabidopsis and rice, in response to salt stress (Kawasaki et al., 2001; Seki et al., 2002). Compared with these model plants, barley, a major crop plant, is a moderate salt-tolerant species and thus is a good target with which to study the mechanisms of salt tolerance in crop plants. However, until now, only one paper has reported using barley cDNA microarrays for transcriptome analysis (Öztürk et al., 2002). A customized cDNA microarray was prepared previously using 460 kinds of barley salt-responsive genes obtained by differential display under long-term salt-stress conditions, and the transcriptomes in leaves and roots were investigated during the initial phase of salt stress (A Ueda et al., unpublished data).
Generally, salt stress causes both osmotic stress and ionic stress. Under salt stress, osmotic stress is triggered by an excess of salt in the soil, and ionic stress is caused by the over-accumulation of salt in the cells. These stresses individually affect the physiological status (Lefèvre et al., 2001; Ueda et al., 2003). During the initial phase of salt stress, osmotic stress is dominant in the inhibition of plant growth. In this work, in order to distinguish the effects of osmotic stress and ionic stress on gene expression, induced genes were studied in barley leaves and roots during the initial phase of osmotic stress using the barley customized cDNA microarray, previous results on expression profiling under salt stress were compared (A Ueda et al., unpublished data), and then the osmotic stress-specific gene expression was identified.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material and stress treatments
Barley (Hordeum vulgare L. cv. Haruna-nijyo) seedlings were grown hydroponically in half-strength Hoagland solution with doubled iron concentration under 13 h light phase (light intensity 400 µmol m–2 s–1, 25 °C, humidity 70%)/11 h dark phase (22 °C, humidity 75%). Osmotic stress was applied to 15-d-old seedlings with three leaves by Hoagland solution containing polyethylene glycol (PEG, average molecular weight 6000). The final concentration of PEG (approximately 20% w/v) was adjusted with the PotentiaMeter to be osmotically equivalent to that of 200 mM NaCl. Barley leaves and roots were harvested at 1 h and 24 h after the stress treatments. Stressed leaves and roots were stored at –80 °C until RNA extraction.
Measurement of leaf water potential
Changes in leaf water potential under osmotic stress and salt stress were monitored using the PotentiaMeter (Decagon, Pullman, MA). The 2nd leaf blades were used to determine leaf water potential. Leaf blades were cut 3 cm in length, set into the attached chamber, and then sealed with parafilm. Prior to measurement, the leaves were incubated in the chamber for 30 min to equilibrate the ambient water potential at 25 °C.
Monitoring barley transcriptome
The barley cDNA microarray was prepared using 460 salt-responsive genes obtained by differential display (Ueda et al., 2002; T Takabe et al., unpublished data), and included non-plant cDNAs (Array ControlTM, Ambion, Austin, TX) as external standards for normalizing the signal intensities among different slides. The cDNA inserts of 460 clones were amplified by 50 cycles of PCR (94 °C for 1 min, 54 °C for 1 min, and 72 °C for 2 min). PCR was performed in a solution containing 1x PCR buffer, 0.2 mM dNTPs, 250 nM M13 forward and reverse primers, 200 ng of plasmid DNA, and 2.5 U of Taq polymerase (Perkin-Elmer Life Science, Boston, MA). After ethanol precipitation, the concentration of PCR products was adjusted to between 150 ng µl–1 and 250 ng µl–1 in 50% (v/v) dimethylsulphoxide. DNA was spotted in quadruplicate on aminosilane-coated slides (GAPSII Coated Slide; Corning, Acton, NY) using the GeneTAC G3 arrayer (Genomic Solutions, Ann Arbor, MI). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). Cy5- (control) and Cy3- (stressed) labelled cDNAs were synthesized by reverse transcribing 75 µg total RNA using MICROMAX Direct Labeling Kit (Perkin-Elmer Life Science). Hybridization was carried out using the automated GeneTAC Hybridization station (Genomic Solutions) for 16 h at 60 °C for barley targets and 55 °C for rice targets. Hybridized slides were washed in 1x SSC, 0.2% SDS at 55 °C for 20 s twice, in 0.1x SSC, 0.2% SDS at 55 °C for 20 s twice, and finally in 0.1x SSC at 25 °C for 20 s twice. Each hybridization was repeated at least three times (technical replicates) using RNA from at least three biological replicates. After hybridization, the slides were scanned using GeneTACTM LS IV laser scanner and analysed using the Integrator Analyzer 3.3 software (Genomic Solutions). The signal intensities of elements in each slide were normalized globally. The significance of differential regulation was measured statistically by the ANOVA F-test, available in the SAS package.
Northern blot analysis and real-time RT-PCR
Differential regulation of selected genes was validated by performing northern blot analysis as described previously (Ueda et al., 2001). For real-time RT-PCR analysis, first strand cDNA was synthesized from 5 µg total RNA using SuperScriptIII reverse transcriptase (Invitrogen). Quantitative PCR was performed with LightCycler using the LightCycler-FastStart DNA Master SYBR Green I kit (Roche, Basel, Switzerland). All PCR experiments were performed with 3 mM MgCl2. Specific primers were as follows: Actin forward (5'-AGACCTTCAACACCCCTGCTATGT-3') and reverse (5'-CCAATCCAGACACTGTACTTCCTT-3'); GAPDH forward (5'-TTTCGGAAGGATCGGGAG-3') and reverse (5'-ATCAGGTCGACAACACGGTT-3'); phosphogluconate dehydrogenase forward (5'-ATTATCCGGGCAAGGTTTCTT-3') and reverse (5'-CCATAGAACCTGAAGCTACA-3'). Fold changes were estimated by the expression value of actin as an internal standard and transferred to log2 ratio (osmotic stress/control). Real-time RT-PCR experiments were repeated using total RNA from three biological replicates.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Physiological status of barley under osmotic stress
Under salt or drought stress, plant tissues are severely dehydrated, with a consequent decrease in leaf water potential. Hence, leaf water potential is often used as a parameter of water stress (Ueda et al., 2003
). To compare the physiological status of barley under osmotic stress and salt stress, the changes in leaf water potential were monitored after both treatments. By 1 h of PEG treatment (approximately 20% w/v), leaf water potential declined from –0.52 to –0.89 MPa (Fig. 1). Although the lowest value was observed at 10 h (–1.45 MPa), leaf water potential was restored to –0.78 MPa after 24 h of stress treatment. The barley plants were also subjected to salt stress (200 mM NaCl) which was the same osmotic potential as the PEG solution. During the first 10 h, a similar decrease in leaf water potential was observed under osmotic and salt stress conditions (Fig. 1). This indicated that barley plants suffered from the same degree of water stress under either PEG or salt stress during the first 10 h. However, recovery was greater under osmotic stress than under salt stress after 24 h of treatment, suggesting Na+ and/or Cl– may exert additional osmotic effects.
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Identification of osmotic stress responsive candidates
Four hundred and sixty non-redundant salt-responsive ESTs were arrayed on glass slides and changes in their abundance in response to osmotic stress were monitored. Fifty-two genes showed differential expression under osmotic stress during the first 24 h (Table 1). During the same period, however, 92 genes were differentially expressed under salt stress (A Ueda et al., unpublished results). As seen in Fig. 2, transcript levels of 18 of the up-regulated genes under salt stress were also up-regulated under osmotic stress. Sixteen genes also showed down-regulation under both osmotic and salt stress conditions. However, a total of 18 genes showed different expression patterns under osmotic stress than under salt stress (Table 1). Based on the mode of regulation and the stimulus to which they responded, differentially expressed genes were classified into six groups (Table 1).
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Validation of microarray experiments
The microarray data were validated by performing northern blot analysis for a few randomly selected genes. As seen in Fig. 3, the mRNA abundance of genes encoding PMP3 and PRP (proline rich proteins) was higher under salt and osmotic stresses, (although the effect was much larger with NaCl than with PEG). The transcript level of phospholipase C in roots was down-regulated by both stress treatments (Fig. 3). These expressions were similarly regulated by osmotic and salt stresses. Up-regulation of hypothetical protein, a candidate in Group 3 in Table 1, was confirmed by northern blot analysis. On the other hand, expression of the no homologue protein gene was up-regulated under salt stress, but not osmotic stress. In addition, the microarray data were also evaluated by real-time RT-PCR (Table 2). The expression levels of GAPDH (one of the up-regulated candidates) and phosphogluconate dehydrogenase (one of the down-regulated candidates) were examined by real-time RT-PCR in osmotically stressed-barley roots. The results for real-time RT-PCR analyses showed that fold changes of both GAPDH and phosphogluconate dehydrogenase were also identical to the microarray data.
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Information on the transcriptome in barley under drought stress was reported by Öztürk et al. (2002)
. Drought stress and PEG-mediated osmotic stress cause dehydration from plant tissues, but they are essentially different type of stresses. Notwithstanding, some of the typical stress responsive genes, such as P5CS, lipoxygenase, and aldehyde dehydrogenase, were up-regulated in both studies. Interestingly, they reported the up-regulation of a kind of water channel gene (WCP-IV) in barley roots after 10 h of drought stress (Öztürk et al., 2002). This study's array carried two genes for water channels in barley, and neither gene was up-regulated under either osmotic or salt stress. Water permeability is one of the important determinants for stress tolerance in plants (Aharon et al., 2003), therefore, further study including the analysis of tissue-specific expression should provide useful information.
Crosstalk between salt and osmotic signals under salt stress
Among the 62 genes that showed up-regulation under salt stress (A Ueda et al., unpublished results), 18 were up-regulated by osmotic and salt stress conditions (Fig. 2). Sixteen of the 30 salt-repressed genes showed down-regulation under both the stress treatments. However, even among these groups, there were many differences in location, or timing, or extent of the induction; and only four of the up-regulated and six of the down-regulated genes had a truly similar pattern under both stress conditions. The others, and especially the genes listed in Groups 3, 4, 5, and 6, were subject to differential regulation due possibly to ionic and/or other secondary stresses caused by salt. It is likely that common regulatory networks and/or signalling intermediates govern the expression of genes that are regulated equally by both the stress stimulus (Shinozaki and Yamaguchi-Shinozaki, 1998). The magnitude of expression of such genes was similar under both the stress conditions with the exception of genes encoding cytochrome P450 and PRP. This result suggests that these genes are regulated by the two stress cues. Further understanding of such interactions should become feasible with the availability of regulatory sequences of genes. Nevertheless, the diversity in biological functions of these commonly regulated genes suggests that several of the downstream responses of salt and osmotic stresses may also be shared.
The present study revealed 18 genes (Groups 3–6) that are differentially regulated by only osmotic stress, but not salt stress. This indicates that the changes in expression levels expected from the osmotic component of salt stress, may have been suppressed by other salt-mediated signals. Such differences in transcript profile might reflect the adaptive values of biochemical pathways under different stress conditions. For example, osmotic stress triggered up-regulation of P5CS and down-regulation of the proline transporter in leaf tissues under osmotic stress (Table 1; Groups 1 and 4). The transcript for the proline transporter gene was abundant in the root tip region, especially the root cap and cortex cells (Ueda et al., 2001); and proline made in leaves may be translocated to the root tip region. Co-ordinate regulation was observed in expressions of sucrose synthase and sugar transporter genes in root tissues. Up-regulation of sucrose synthase and down-regulation of the sugar transporter were triggered by osmotic but not salt stress (Table 1; Groups 3 and 4), hence, this would be a good target to dissect further signalling controls for the differentiation of osmotic stress from ionic stress.
In this study, genes were identified that are regulated by osmotic stress under salt stress by comparing the expression profiles of 460 salt-responsive EST genes using microarrays. These results demonstrate that a number of genes that are regulated under salt stress are mediated by osmotic stress caused by excessive salts. A dissection of osmotic stress-mediated responses under salt stress might accelerate the genetic improvement of salt tolerance in target environments.
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Acknowledgements |
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This research was supported by a Grant ‘Research for the future’ to TT and a Research Fellowship of JSPS for Young Scientist to AU. We are greatful to Dr AT Jagendorf (Cornell University) for critical reading of the manuscript and valuable discussions.
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