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a Department of Plant Biology, 190 ERML, University of Illinois, Urbana, IL 61801, USA
b Horticulture Department, Poole Agriculture Center, Box 340375, 50 Cherry Rd., Clemson University, Clemson, SC 29634-0375, USA
* Corresponding author (VBAIRD{at}CLEMSON.EDU)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drought and high salinity are two of the most important environmental stresses that alter plant water status and severely limit plant growth and development, and thus crop productivity. Dehydration causes a number of physiological and biochemical changes in plants, such as a decrease in photochemical activities, reduction of CO2 fixation, accumulation of osmolytes and osmoprotectants, and alteration in carbohydrate metabolism (Tabaeizadeh, 1998). Also, high salinity (e.g., increased concentrations of Na+ and Cl- in the soil solution) causes osmotic/ionic stress (Hasegawa et al., 2000). The transduction pathways for osmotic and other environmental stress responses are likely to be very complicated, and will involve a number of signal molecules such as abscisic acid (ABA), cyclic nucleotides, and inositol polyphosphates. Thus, the precise mechanism(s) by which plants respond to drought or high salinity remains unresolved. However, at the molecular level, most of the changes are likely the result of alterations in the expression of genes. Therefore, it is important to identify the relevant genes and characterize their regulation in response to water and/or salinity stress.
Recently, a number of drought-responsive (Ingram and Bartels, 1996; Kim et al., 2000; Nepomuceno et al., 2000) and salinity-responsive (Moons et al., 1997; Ramani and Apte, 1997; Wei et al., 2000) genes were cloned and characterized from different plant species. Transcription of many of these genes (e.g., those encoding the late-embryogenesis-abundant, LEA proteins, Espelund et al., 1995; rd29A and rd29B, Yamaguchi-Shinozaki and Shinozaki, 1993; glyceraldehyde-3-phosphate dehydrogenase, Jeong et al., 2000; and ABA-responsive element binding proteins AREB1 and AREB2, Uno et al., 2000) is up-regulated by both drought and salinity stress. On the other hand, the expression of other genes is regulated specifically by either drought stress (Jonak et al., 1996) or high salinity (Binzel and Dunlap, 1995; Nemoto and Sasakuma, 2000). In addition, organ-specific expression of a salinity-induced gene (Pgm1) was reported for ice plant, Mesembryanthemum crystallinum L. (Forsthoefel et al., 1995). Despite these studies, relatively little is known about the fundamental differences and cross-talk between drought and high salinity response pathways in plants.
Differential display-polymerase chain reaction (DD-PCR) (Liang and Pardee, 1992) is a simple, sensitive and powerful method for screening cDNAs, and is useful in characterizing tissue-, organ- or development-specific cDNAs (Cushman and Bohnert, 2000). DD-PCR has been used successfully to isolate a number of differentially expressed genes from plants (Martin-Laurent et al., 1997; Roux and Perrot-Rechenmann 1997; Visioli et al., 1997; Deleu et al., 1999; Wei et al., 2000). In the work reported here, 17 cDNA clones were isolated from sunflower by means of DD-PCR. Genes corresponding to 13 of these cDNAs were confirmed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to be expressed differentially in response to osmotic stress. Their expression patterns were analyzed in leaves of drought-stressed plants, and in roots and shoots of drought- or salinity-stressed seedlings.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drought or Salinity Treatments
Analysis of dry-weight comparisons showed that tissue water content decreased 10% after drought treatment for 6 d, air-drying for 10 h, or treating seedlings for 6 h with 250 mM NaCl. Therefore, stress response experiments for analyzing gene expression patterns under conditions of drought or salinity stress were performed as follows.
Drought treatment was accomplished by the modified method of Ouvrard et al. (1996). One-month-old plants were subjected to progressive drought by withholding water. Young, fully expanded leaves were collected daily (for 6 d) for RNA extraction. One-week-old seedlings were also drought stressed by placing them on filter paper and air drying for 10 h. Roots and shoots from stressed seedlings were collected separately for RNA extraction.
For high salinity treatment, 1-wk-old seedlings were transferred to a container of 250 mM NaCl. Seedlings were treated with the salt solution for up to 9 h. Control seedlings were grown in water. Roots and shoots from control and stressed seedlings were collected separately for RNA extraction.
Differential mRNA Display
Total RNA from 1 g of tissue was isolated from treated and control plants with RNAqueous Kit (Ambion Inc, Austin, TX, USA). DNA contaminants were removed with MessageClean Kit (GenHunter Corp., Nashville, TN, USA). The RNA was stored at -70°C. DD-PCR was performed as described in the RNAimage Kit (GenHunter Corp.). For drought-stress experiments, samples were taken over a 6-d period for both treated and control plants. Each reverse transcription (RT) reaction contained 5 mM KCl, 10 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 25 µM of each dNTP, 0.2 µM anchor primer (T11VC, T11A, T11G, or T11C, where V is A, G, or C; GenHunter, Corp.), 0.5 µg total RNA and 100 U MMLV reverse transcriptase. RT reactions were performed at 40°C for 60 min followed by incubation at 70°C for 5 min.
One tenth of the cDNA was used for PCR amplifications in the presence of 2.5 µM of each dNTP, [
-33P]dCTP (0.075 mBq, NEN, Boston, MA, USA), 0.2 µM of the same anchor primer as in the RT reaction, 0.2 µM of an arbitrary primer (from AP1 to AP12, GenHunter Corp.), 0.5 U of Taq DNA polymerase (Promega, Madison, WI, USA) with its own buffer containing 1.5 mM MgCl2. After denaturation at 92°C for 3 min, 40 PCR cycles (i.e., cycle consists 92°C for 45 s, 40°C for 2 min and 70°C for 2 min) were performed and followed by a 5 min extension step at 70°C.
One sixth of the PCR product was mixed with formamide loading buffer (GenHunter Corp.), denatured for 5 min at 95°C, and analyzed on a denaturing 6% (w/v) acrylamide gel. The region(s) of the gel containing the band(s) of interest was excised and eluted into 100 µL of 10 mM Tris pH 8.0, 1 mM EDTA by incubating for 15 min in boiling water bath. The supernatant (5–10 µL) was reamplified under the same PCR conditions except no [-33P]dCTP was added.
For salinity-stress treatment, total RNA was isolated from roots or shoots of stressed and control seedlings over a 9-h period. The anchor primers used were T11A, T11G, and T11C (GenHunter Corp.), and the arbitrary primers were from AP1 to AP24 (GenHunter Corp.). DD-PCR was performed by means of the same protocol as described above. DD-PCR amplification for each primer pair was performed at least twice from RNA samples isolated separately for each time point.
Cloning and Sequencing
The reamplified products were cloned into pGEM-T Easy vector according to the manufacturer's protocol (Promega), and sequenced with T7 and SP6 primers with the ABI prism Dye Terminator kit and ABI model 373 automated DNA sequencer (Perkin Elmer, Branchburg, NJ, USA). The nucleotide sequence or the deduced amino acid sequence of each clone was compared with DNA, EST, and protein sequences from various databases by means of the basic local alignment search tool (BLAST) (Altschul et al., 1990).
Quantitative Reverse Transcriptase Polymerase Chain Reaction
The expression pattern of each clone was further confirmed by quantitative RT-PCR using a gene-specific primer pair based on the nucleotide sequence of each clone. The sunflower gene-specific primer pairs used for quantitative RT-PCR are listed in Table 1. The primer pair for amplification of plant 18S rRNA (as the internal standard) and the 18S rRNA inhibitory competitive primer pair were from the QuantumRNA kit (Ambion). Each RT reaction was performed as described in the Differential mRNA Display section (above), except the primers were random hexamers (Promega, Madison, WI, USA). For each PCR reaction, one tenth of the cDNA was added in a mixture containing 100 µM of each dNTP, 0.2 µL of [-32P]dCTP (0.075 mBq, NEN), 1 µM of each gene-specific primer pair, 1 µM of 18S rRNA primer pair and 18S rRNA inhibitory primer pair mixture (2:8), 0.5 U of Taq DNA polymerase (Promega) with its own buffer containing 1.5 mM MgCl2. After denaturation at 95°C for 3 min, 20 PCR cycles (95°C for 30 s, 60°C for 30 s, and 72°C for 30 s) were performed, followed by a 1-min extension step at 72°C. One-fifth of the PCR product was mixed with formamide loading buffer, denatured for 5 min at 95°C, and analyzed on a denaturing 6% acrylamide gel.
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RESULTS |
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Except for the gene corresponding CAp1-2U, which was expressed constitutively (Fig. 3), the other four genes corresponding to drought-regulated clones were found to have similar expression patterns to those identified from the original differential display gels. Furthermore, quantitative RT-PCR basically confirmed the differential expression of nine out of the twelve salinity-regulated clones (data not shown).
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Gene Expression Patterns under Drought- and Salinity-Stress Conditions
For the 13 clones confirmed to be differentially expressed in response to either drought or high salinity, differences and similarities in their stress-regulated expression were investigated further. Quantitative RT-PCR was again used to investigate expression of each clone in roots and shoots of drought- or salinity-treated seedlings, and in leaves of drought-treated plants. The results are shown in Fig. 4 and summarized in Table 3.
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Transcripts from RSC5-U, RSG11-U and RSG15-U were not detected in untreated, control samples. However, their expression was detected in salinity-treated and in drought-treated seedlings, and these genes were highly expressed in leaves taken from plants following drought treatment (Fig. 4, Table 3). Expression of SA3-U was up-regulated in drought-treated leaves and drought- or salinity-treated seedling shoots, but downregulated in seedling roots by drought or high salinity (Fig. 4, Table 3). SC7-U was constitutively expressed at a high level in seedling shoots of both control and salinity-treated plants. However, its expression was up-regulated in seedling roots by salinity or drought treatments, but downregulated in seedling shoots and leaves by drought (Fig. 4, Table 3). In both control and drought-treated plants, RSC1-U was poorly expressed in seedling roots, but highly expressed in leaves. In addition, its expression was modestly up-regulated in seedling roots by salinity and in seedling shoots by exposure to drought and salinity (Fig. 4, Table 3). Expression of SG2-U was up-regulated by both types of abiotic stress in all organs examined, and expression of RSG22-D was found to be downregulated by both stresses in all organs examined (Fig. 4, Table 3).
Differential effects between drought and salinity stress were observed when expression patterns of genes corresponding to RSG10-U were analyzed. RSG10-U was found to be up-regulated in seedling roots and downregulated in seedling shoots in response to exposure to high salinity. Under drought conditions, expression of this gene was downregulated in seedling roots, but up-regulated in seedling shoots and leaves (Fig. 4, Table 3).
On a comparative basis, for analyzing expression of genes corresponding to the four drought-regulated clones, the control 18S rRNA primer pair amplified a single, 315-bp fragment at the identical concentrations from the first strand cDNAs produced from both treated and control leaves. Similarly, for the nine genes corresponding to high salinity-regulated clones, the 18S rRNA control primers amplified a 315-bp fragment to identical concentrations from cDNAs produced from roots and shoots of treated and control seedlings (Fig. 4).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern gel blot analysis along with detailed quantitative RT-PCR confirmed that the genes corresponding to 13 of the original 17 cDNA clones were differentially expressed in response to drought and/or salinity stress. Interestingly, their individual expression patterns were found to differ in response to drought and/or salinity stress in an organ-specific manner. Even those genes whose expression was up-regulated or downregulated by both high salinity and drought were expressed at different levels under individual stress conditions. It has been proposed that high-throughput stress-specific gene expression analysis is important for understanding gene function (Cushman and Bohnert, 2000). The results reported here suggest that organ- or tissue-specific, as well as stress-specific gene expression analyses are necessary in gene identification and characterization studies.
Although the expression of a number of genes was shown to be enhanced by exposure to both drought and salinity stress (Claes et al., 1990; Espelund et al., 1995; Jeong et al., 2000; Uno et al., 2000), differential responses to dehydration and high salt concentration have been documented in only a few studies. The expression of the glucose-6-phosphate dehydrogenase (G6PDH) gene in wheat (Nemoto and Sasakuma, 2000) and the 70-kDa (catalytic) subunit of tonoplast H+-ATPase gene in tomato (Binzel and Dunlap, 1995) is induced specifically by NaCl, but not drought. Furthermore, the expression of a salinity-induced gene (Pgm1) from ice plant is up-regulated in leaves by both drought and high salinity, not affected in roots by salinity-stress, and downregulated there by drought-stress (Forsthoefel et al., 1995). A mitogen-activated protein kinase gene (p44MMK4) was expressed under drought or cold stress, but not in response to high salinity (Jonak et al., 1996).
The 13 partial cDNAs reported here can be organized into three groups on the basis of their different expression patterns in response to osmotic stress. The first pattern (Pattern I, Table 3) is illustrated by genes whose expression is either exclusively up- or downregulated in all organs in response to both stresses (i.e., clones RSC1-U, RSC5-U, SG2-U, RSG11-U, RSG15-U, RSG22-D, and GAp1-D), or whose expression is up- or downregulated in an organ-specific manner (i.e., clones SA3-U and SC7-U). The expression of CAp1-1U and VC2-D, which represents the second pattern (Pattern II, Table 3), was up- or downregulated by drought stress only, and not affected by salinity. The third pattern (Pattern III, Table 3) demonstrates a differential effect between drought and salinity stress and between organs. Pattern III was observed when characterizing the expression of the gene corresponding to RSG10-U. For this pattern, expression is up- or downregulated depending on the organ and/or the stress.
Response to osmotic stress stimuli is very likely mediated by one or more signal transduction pathways. In vegetative tissues, endogenous ABA levels increase in response to dehydration (Zeevaart and Creelman, 1988) or by exposure to high salinity (Moons et al., 1997). Recent studies on the promoters of drought- and ABA-responsive genes suggest that both ABA-independent and -dependent signal pathways are involved in the dehydration response of plant cells (Shinozaki and Yamaguchi-Shinozaki, 2000). It is interesting that the expression of genes [i.e., Lea (Espelund et al., 1995) and rd29A and rd29B (Yamaguchi-Shinozaki and Shinozaki, 1993)] that can be induced by both drought stress and by salinity stress, are also induced by ABA. However, other drought- or salinity-stress-specific responsive genes are expressed in an ABA-independent manner (Binzel and Dunlap, 1995; Forsthoefel et al., 1995; Jonak et al., 1996; Nemoto and Sasakuma, 2000). Therefore, understanding how the 13 genes reported here respond to exogenous ABA is important for a more complete understanding of the drought-stress, salinity-stress and ABA-responsive pathways. Our analyses showed that transcription of genes with expression pattern I (i.e., clones RSC1-U, RSC5-U and RSG11-U) was up-regulated by exogenous ABA in both seedling roots and shoots, but transcription of genes with expression Patterns II or III (i.e., clones VC2-D, CAp1-1U and RSG10-U) was not affected by ABA treatment (Liu, 2002). These results suggest that plants respond to drought or salinity stress by different pathways, and cross-talk between both stresses is mediated through an ABA-responsive pathway.
VC2-D is homologous to the lytB genes from both plants and bacteria (Cunningham et al., 2000; Gustafson et al., 1993; Wosten et al., 1997; Ham et al., 1999; Lopez et al., 2000). In those studies involving bacteria, lytB was found to be related to nutritional- and temperature-stress response, and it was reported to encode a pneumococcal murein hydrolase, which plays an important role in cell division (Wosten et al., 1997). In tobacco (Nicotiana spp.), a lytB homolog was expressed constitutively in various organs, and at an increased level in response to viral (CaMV) infection. It was suggested that lytB is a novel host factor interacting with the viral coat protein CMV2b (Ham et al., 1999). Recently, Cunningham et al. (2000), working with A. aestivalis, proposed that lytB encodes an enzyme of the deoxyxylulose-5-phosphate pathway, which catalyzes a step at or subsequent to the branch point to form isopentenyl diphosphate and dimethylallyl diphosphate. Transformation of a Synechocystis lytB gene and a lytB gene from A. aestivalis each enhanced accumulation of carotenoids in Escherichia coli (Cunningham et al., 2000). The function of the lytB gene product in plant cells exposed to abiotic stress is unknown, and our finding, that expression of the lytB-like gene was specifically downregulated, suggests that its role in stress response is likely to be complex.
CAp1-1U shows a high level of homology to a tobacco guanylate kinase (GK) gene (Kumar, 2000), which encodes an enzyme critical to the biosynthesis of nucleotides. Guanylate kinase (ATP:GMP phosphotransferase, EC2.7.4.8) catalyzes the reaction (d)GMP + ATP 
(d)GDP + ADP (Miech and Parks, 1965). This step is very important in the recovery of cyclic-GMP, and the balance of ATP and GTP concentrations within the cell. Therefore, GK is thought to be an important enzyme that is fundamental to second-messenger signal transduction pathways (Brady et al., 1996). Recently, genes for GK have been preliminarily characterized from three plant species (i.e., Arabidopsis, Kumar et al., 2000; lily and tobacco, Kumar, 2000). AGK-1 and AGK-2, were shown to be constitutively expressed in all tissues of Arabidopsis, but their transcription level is highest in roots. However, nothing is known about expression of GK in response to environmental stresses. In the work reported here for sunflower, expression of the GK-like gene corresponding to CAp1-1U was up-regulated in drought-stressed leaves, seedling roots and seedling shoots, but not in those same organs when plants were exposed to high salinity. This implies that the expression of GK may be specifically related to signaling the onset and/or immediate effects of water deficit throughout the plant.
Expression of the gene corresponding to GAp1-D was downregulated by both stress conditions in all organs examined. GAp1-D was found to be highly homologous to a selenium-binding protein from Arabidopsis. Selenium (Se) plays an important role in the growth and development of mammals. Selenium binding protein (SBP) is reported to be involved in mediating the anti-carcinogenic effects of Se, and it is expressed differentially in various organs and cell lines (Lanfear et al., 1993; Yang and Sytkowski, 1998). The function of a SBP in plants is unknown. Recently, a SBP gene was obtained from ESTs of a moss treated with exogenous ABA (Machuka et al., 1999). In tall fescue (Festuca arundinacea Schreber), uptake and accumulation of Se is affected by soil moisture (Tennant and Wu, 2000). These recent results, together with our findings, show that a SBP may be important in regulating the Se concentration of plant cells in response to environmental stresses.
Transposable elements, which can cause genetic variation and alter gene expression, are important in host adaptations to environmental changes (Kunze et al., 1997). Expression of plant retrotransposons has been reported to be activated at the transcriptional level in response to different biotic and abiotic stresses (Grandbastien, 1998). For example, expression of the Tnt1 and Tto1 retrotransposons is induced by wounding, methyl jasmonate, CuCl2 and salicylic acid (Kumar and Bennetzen, 1999). However, activation of transposable elements by drought- and/or salinity-stress has not been clearly documented. Here we report on two partial cDNA clones, similar to two different types of transposable elements, that represent genes differentially expressed in response to drought and/or salinity stress. Clone CAp2-U shows homology to the activator-like transposable element of Pennisetum glaucum (L.) R. Brown, and clone RSG10-U is orthologous to a polyprotein (reverse transcriptase) of S. bicolor (Fig. 2D and 2E). Further characterization of these two genes will provide information on the role of transposable elements in the host plant's adaptation or response to environmental stress.
Cloning and characterization of full-length cDNAs and promoter regions of the genomic sequences corresponding to the stress-regulated clones reported here will be necessary to fully understand the response mechanism(s) of plant cells to different environmental stresses. Such studies should identify common and/or unique regulatory elements, and thus provide insight into the mechanism of a gene's individual expression, as well as its potential role in stress response. This information will in turn help us to understand better signaling and interactions between the major osmotic stress-response pathways.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication July 10, 2002.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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