This Article Abstract Full Text (PDF) All Versions of this Article: 133/3/1397    most recent pp.103.028019v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (27) Citing Articles via Google Scholar Google Scholar Articles by Ekman, D. R. Articles by Dean, J. F.D. Search for Related Content PubMed PubMed Citation Articles by Ekman, D. R. Articles by Dean, J. F.D. Agricola Articles by Ekman, D. R. Articles by Dean, J. F.D.

GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

SAGE Analysis of Transcriptome Responses in Arabidopsis Roots Exposed to 2,4,6-Trinitrotoluene¹

Department of Biochemistry and Molecular Biology (D.R.E., W.W.L., A.E.P., J.F.D.D.) and Daniel B. Warnell School of Forest Resources (W.W.L., J.F.D.D.), University of Georgia, Athens, Georgia 30602; and National Exposure Research Laboratory, Ecosystems Research Division, United States Environmental Protection Agency, Athens, Georgia 30605 (N.L.W.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Serial analysis of gene expression was used to profile transcript levels in Arabidopsis roots and assess their responses to 2,4,6-trinitrotoluene (TNT) exposure. SAGE libraries representing control and TNT-exposed seedling root transcripts were constructed, and each was sequenced to a depth of roughly 32,000 tags. More than 19,000 unique tags were identified overall. The second most highly induced tag (27-fold increase) represented a glutathione S-transferase. Cytochrome P450 enzymes, as well as an ABC transporter and a probable nitroreductase, were highly induced by TNT exposure. Analyses also revealed an oxidative stress response upon TNT exposure. Although some increases were anticipated in light of current models for xenobiotic metabolism in plants, evidence for unsuspected conjugation pathways was also noted. Identifying transcriptome-level responses to TNT exposure will better define the metabolic pathways plants use to detoxify this xenobiotic compound, which should help improve phytoremediation strategies directed at TNT and other nitroaromatic compounds.

View larger version (12K): [in this window] [in a new window]   Figure 1. Chemical structures of nitroaromatic and heterocyclic nitroamine explosives. The nitroaromatic explosives of greatest concern for environmental contamination, TNT, RDX (Royal Demolition Explosive), and HMX (High Melting Explosive), are depicted.

To obtain a more complete picture of the metabolic processes plants employ to cope with nitroaromatic agents, serial analysis of gene expression (SAGE; Velculescu et al., 1995, 1997) was used to identify transcriptome-level responses in Arabidopsis seedling roots exposed to TNT. Although this technique has been widely used to study gene expression changes in human cancers (Riggins and Strausberg, 2001), only two published reports describe the use of SAGE in plants: a study of gene expression in rice (Oryza sativa) seedlings (Matsumura et al., 1999) and an examination of wood formation in loblolly pine (Lorenz and Dean, 2002). The identification of plant genes and pathways responding to TNT exposure at the transcriptional level should facilitate a more reasoned approach to the development of plants better suited for use in phytoremediation of TNT-contaminated sites.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

SAGE Library Characterization and Comparison of Tags to the Arabidopsis Genome

SAGE libraries representing transcripts expressed in Arabidopsis root tissues grown in the presence (15 mg L^–1) or absence of TNT were sequenced to characterize about 30,000 tags from each. The data sets from these analyses are available through the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). Of the 32,203 tags characterized from the library for TNT-treated tissue, 12,005 represented unique transcripts, but 7,900 of these were singletons. Similarly, 12,721 unique tags were encountered among the 32,104 characterized in the control library, with 8,322 of these representing singletons. A double-reciprocal plot of the total unique tags identified versus the total number of tags sequenced after each sequencing run was used to estimate the rate of transcript discovery (Fig. 2). From this, transcriptome sizes of approximately 21,000 and 15,000 were estimated for control and TNT-treated root tissues, respectively. Only 25% of all unique tags (5,084 of 19,640) were detected in both libraries.

View larger version (39K): [in this window] [in a new window]   Figure 2. Estimates of transcriptome size in Arabidopsis roots. Double-reciprocal plots of new tag discovery versus total tags sequenced are shown for the TNT-treated (top) and control (bottom) root libraries. The estimated transcriptome size was taken as the 1/y intercept from the linear regression depicted in each graph.

Of the 1,045 most abundant tags in both SAGE libraries (which included all tags seen 10 or more times among the 64,176 tags characterized) about 70% (739), could be matched to a single model gene in the Arabidopsis Genome Initiative (AGI) database. Most of the remaining tags (233) matched sequences found one or more times among all Arabidopsis sequences deposited in GenBank. Many such tags were found in expressed sequence tags (ESTs) but were not positioned adjacent to the CATG sequence closest to the 3' end of the transcript. This suggests that they might have been generated from alternatively spliced transcripts. A good example of this was the fourth most abundant tag in the control tissue library (AGGTCTTGGT, counted 134 times). This sequence appears only one time in the Arabidopsis genome and falls immediately 3' of the penultimate CATG site on the annotated transcript of gene At3g09260 (-glucosidase). The full-length transcript for this gene annotated in the AGI database corresponded to the second most abundant tag in the control library (ATTTGCCAGA, counted 286 times) and the fourth most abundant tag in the TNT treatment library (counted 259 times).

Of the remaining unidentified tags in the top 1,045, one that did not correspond to anything listed in the AGI database (AGTAACGATA) matched a sequence found on the Arabidopsis mitochondrial genome, and numerous ESTs incorporating this sequence have been deposited in GenBank. About 5% of the tags (57) matched more than one model gene, whereas a few tags (14) contained at their 3' end a contiguous stretch of "A" residues, suggesting that they incorporated part of a poly(A) tail. Two tags, one of which (TCCCCGTACA) was the 37th most abundant tag overall, could not be matched to any Arabidopsis sequences in GenBank.

Identification of Differentially Expressed Transcripts

TNT treatment induced an apparent increase of at least 5-fold in 242 tags, whereas 287 tags decreased in abundance at least 5-fold in response to this treatment. For tags that were relatively abundant in one library but not observed in the other, a minimum change in expression level was estimated by assuming that a single copy of the tag was found in the library from which it was absent. Several of the tags displaying the greatest induction in response to TNT exposure represented gene products known to be involved in plant responses to oxidative stress, such as monodehydroascorbate reductase and glutathione (GSH)-dependent dehydroascorbate reductase (Table I). The identity of the tag most highly induced by TNT remains uncertain. Its sequence appears twice on chromosome 5, and in one of these instances, the tag lies in the middle of a computationally predicted transcript that has never been isolated. However, a third occurrence of the sequence resides on chromosome 2 at the penultimate CATG site of the transcript for LKP2 (At2g18915), a signaling protein involved in the Arabidopsis circadian clock (Schultz et al., 2001). The evidence suggests the most likely source of this tag to be a splice variant of the LKP2 transcript. In contrast, the second most highly induced tag, which increased nearly 28-fold in TNT-treated roots, definitely represented a glutathione S-transferase (GST). Several of the induced tags represented cytochrome P450s, a large family of enzymes used by plants and other organisms in the detoxification of xenobiotic compounds (Table II). As further indication of the stress imposed upon the seedlings by TNT exposure, tags for various transporter proteins, transcription factors, signal cascade proteins, and heat shock proteins were among those most highly induced by TNT exposure. Other strongly induced tags representing gene products known to be involved in detoxification reactions or in the protection of plant cells against oxidative stress are noted in Table III. Prominent among the enzymes likely to protect against oxidative stress are quinone reductase, ascorbate peroxidase, peptide Met sulfoxide reductase, phospholipid-hydroperoxide GSH peroxidase, and GSH reductase.

Tags that showed the greatest decrease upon exposure to TNT are listed in Table IV. The largest effect was seen for a tag representing a lipid transfer protein (LTP) family member (At4g12550) that has been associated with lateral root formation in response to auxin (AIR1A; Neuteboom et al., 1999). An overlapping transcript of this gene (At4g12445, AIR1B) represented by a different SAGE tag shows the fourth most precipitous decline in response to TNT, whereas a related member of the LTP family (At4g12520, pEARLI-1 homolog) was also severely repressed in response to the TNT treatment (Table IV). TNT exposure also depressed levels of tags representing three major latex protein homologs, another group of widely distributed proteins whose functions are as yet unknown (Stromvik et al., 1999). Other depressed tags included those for various transcription factors, membrane channels, cytoskeletal elements, and ribosomal proteins.

Validation of SAGE Results Using Quantitative PCR

SAGE has previously been shown to provide an accurate reflection of gene expression levels for medium- and high-abundance transcripts (Evans et al., 2002), but the induction or repression of selected Arabidopsis transcripts in response to TNT exposure was independently verified in this study using real-time quantitative PCR. Genes that SAGE analysis suggested were induced (At3g28740; cytochrome P450), repressed (At2g36830; tonoplast intrinsic protein), or remained unaffected (At2g39460; 60S ribosomal protein L23A) by TNT exposure were tested. As shown in Table V, the quantitative PCR data showed general agreement with the SAGE data. With respect to apparent discrepancies between the values determined by the two techniques, it should be noted that the value determined by quantitative PCR is a logarithmic, rather than a linear function of the starting number of templates present. Thus, ratios of expression differences are not generated by simple division of threshold cycle (C_t) values, and even slight differences in the PCR reaction conditions can have strong impact on the C_t values. With respect to SAGE, due to sampling effects, differential expression can be strongly affected by the extent to which sequencing is carried out. Thus, the SAGE-determined value for P450 (At3g28740) induction is significantly underestimated because of the constraints placed on the depth of sequencing in the SAGE libraries. These factors are likely the most significant sources for the slight discrepancies noted in the data produced by the two techniques.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
A major goal of this study was to identify plant-specific enzymes and metabolic pathways that might previously have been overlooked for their importance in conferring tolerance to TNT. To avoid confusion from microbial influences and to minimize variation resulting from environmental fluctuations, this study used root tissues from seedlings grown in sterile liquid culture. The tolerance of Arabidopsis to TNT under these conditions was comparable with that seen in other plants species grown under sterile culture conditions (Hannink et al., 2001). For example, TNT was shown to induce chlorosis and cell death in cultures of Anabena sp. at a concentration of 10 mg L^–1 (Pavlostathis and Jackson, 1999), whereas cell suspension cultures of Datura innoxia were capable of tolerating TNT at up to 29 mg L^–1 without an effect on growth (Lucero et al., 1999). Although the liquid culture system used in this study exposed all portions of the Arabidopsis seedlings to TNT, only the root tissues were sampled for SAGE analyses because under conditions of normal terrestrial growth TNT and its derivatives tend to remain associated with root tissues (Cataldo et al., 1989). Thus, efforts to improve plant tolerance to TNT would seem most likely to benefit from detailed study of the metabolic responses in these tissues.

SAGE Analysis of the Arabidopsis Transcriptome

The Arabidopsis genome has been estimated to contain approximately 25,500 genes (Arabidopsis Genome Initiative, 2000), but given the possibilities for alternative splicing and polyadenylation during transcript maturation, the transcriptional space of Arabidopsis should be substantially larger. This inference was supported by SAGE results identifying more than 19,000 unique tags in our sampling of a mere 60,000 tags from root tissues alone. Just as was seen in a similarly sized SAGE study of rat fibroblast cells (Madden et al., 1997), the discovery of new tags in the Arabidopsis SAGE libraries had not yet begun to level off at this depth of sampling, and estimates suggested an eventual transcriptome size in the range of 35,000 to 40,000 for the root tissues. However, as noted by Velculescu et al. (1999), to obtain a reasonably complete sampling of a somewhat larger transcriptome (approximately 56,000) using SAGE, one would need to sequence on the order of approximately 650,000 tags. Thus, our data likely represent a low estimate of transcriptome complexity in Arabidopsis.

The size of this SAGE study was similar to a hypothetical situation (15,720 genes; 62,178 sampled tags) modeled by Stöllberg et al. (2000) to estimate the maximum likelihood of generating accurate transcript number and transcript copy frequency given different levels of randomness and non-uniqueness in the transcriptome. Modeling suggested that in a study of this size 1.5% to 6% of SAGE tags should represent two or more genes depending on the non-randomness of sequences represented in the transcriptome. This value matched closely with the 5% figure noted for the 1,045 most abundant tags in this study. However, the modeling study did not take into account the further complexity that might be introduced by variations in transcript maturation processes, such as alternative splicing. Adding further uncertainty to estimates of the Arabidopsis root transcriptome size was the observation that among tags categorized as having ambiguous origin, several could only be matched to antisense sequences for transcripts having ESTs on deposit in GenBank (e.g. CACTTGGATT, the 345th most abundant tag, and TCCGAATCAA, the 385th most abundant tag). SAGE previously demonstrated that antisense transcripts for certain genes were abundant in Caenorhabditis elegans (Jones et al., 2001) and Plasmodium falciparum (Patankar et al., 2001), and although not verified in this study, SAGE tags from antisense transcripts have been verified by reverse transcriptase (RT)-PCR in stressed Arabidopsis plants (G. May, personal communication). Naturally occurring anti-sense transcripts have been seen previously in plants and animals (Terryn and Rouzé, 2000), but it is unclear whether the transcripts identified in our study were somehow involved in posttranscriptional regulation of gene expression via RNAi (Vaistij et al., 2002), or perhaps represented non-coding RNAs that serve some other function in plants (MacIntosh et al., 2001).

Multiphase Mechanisms of TNT Detoxification in Arabidopsis

Studies of the mechanisms plants use to metabolize herbicides and other xenobiotics have previously pointed to a multiphase process for detoxification (Ishikawa, 1992; Kreuz et al., 1996; Ishikawa et al., 1997). Following entrance of the compound into the cell, transformation reactions introduce chemical substituents amenable to conjugation; for example, hydroxyl groups added via enzyme-catalyzed oxidation reactions involving cytochrome P450 enzymes and other mixed function oxidases. The modified compound is subsequently conjugated to GSH or any of several six-carbon sugars through the action of GSTs or UDP:glucosyltransferases, respectively. In the third phase of the process, conjugates are sequestered in the vacuole, where they may be inactivated by further modifications or enter into degradation pathways, or are secreted into the apoplasm where they may be covalently coupled into the cell wall.

SAGE analysis indicates that this multiphase process also functions in the metabolism of TNT by Arabidopsis. Although this has been suggested previously for two other plants, Catharanthus roseus and Myriophyllum aquaticum, based on mass-balance studies of TNT disappearance and metabolite production in axenic cultures (Bhadra et al., 1999a, 1999b), the enzymes most likely involved in the process have not previously been determined. The apparent induction of several cytochrome P450 genes by TNT exposure supports oxidation as the initial transformation step in TNT metabolism. Oxidative transformation of TNT in plants has been suggested from analyses of metabolic products (Bhadra et al., 1999b), but reductive metabolites, such as 2-amino-4,6-dinitrotoluene or 4-amino-2,6-dinitrotoluene, have been recovered most often in mass-balance studies of plant cultures exposed to ¹⁴C-TNT (Lucero et al., 1999; Pavlostathis and Jackson, 1999). Typically, however, the fraction of TNT derivatives recoverable from such studies as small molecules has been only a small percentage of the added TNT, and most of the recovered isotopic label remained in unidentified conjugates or bound to high M_r materials. This suggests that after oxidation of TNT, subsequent conjugation reactions occur too quickly to allow for routine measure of the oxidized intermediates.

While hydroxylation is an effective means of modifying xenobiotic compounds for subsequent conjugation, it rarely serves to decrease toxicity of the parent compound and can sometimes make the compound more toxic. In such cases, hydroxylating enzymes must be coordinately expressed with conjugating enzymes that will quickly decrease the toxic nature of the derivatives. The SAGE data suggest that GSTs were the enzymes shouldering primary responsibility for conjugation reactions involving TNT metabolites. Although previous studies have not provided evidence for the conjugation of GSH to TNT, its conjugation to herbicides and other phytotoxins is well known. In fact, detoxification of herbicides by GSH conjugation is used advantageously in agriculture where compounds that elevate GSH and GST levels, known as "safeners," are applied to crops before herbicide application (Davies and Caseley, 1999). Many safeners are aromatic compounds with nitrogen-containing functional groups, not unlike TNT, and their metabolism in plants has been relatively well studied. Enhanced glucosylation reactions in response to safener treatment have also been noted (Kreuz et al., 1991) and may explain the 5-fold induction by TNT of a SAGE tag representing Suc-UDP glucosyltransferase (TCTCGAACCT, At5g20830). From these observations it would seem that many of the mechanisms known to function in safener metabolism may be directly applicable in understanding the response of Arabidopsis to TNT exposure.

On the other hand, the 7-fold induction of anthranilate N-benzoyltransferase (Table III), an enzyme that catalyzes the first committed step of a phytoalexin biosynthetic pathway and that has been shown to be adept at using hydroxycinnamoyl-CoA esters to modify anthranilate (Yang et al., 1997), may suggest a conjugation pathway for TNT derivatives that has not been previously suspected. Bhadra et al. (1999b) found that oxidation of the methyl group converted nearly 20% of the TNT added to plant cultures to various benzoyl derivatives. Because benzoate:CoA ligase is a member of an enzyme family that includes 4-coumarate:CoA ligase (Beuerle and Pichersky, 2002), the near 3-fold induction of 4-coumarate:CoA ligase seen in the TNT-treated roots may reflect a mechanism for activating oxidized TNT derivatives before anthranilate conjugation.

Conjugation of GSH with cellular toxins is generally thought to confer increased solubility, decreased toxicity, and increased transport competency on the toxin derivatives (Schröder and Collins, 2002). The transport of GSH conjugates across membranes is an ATP-dependent process handled by ABC transporter proteins (Martinoia et al., 1993; Li et al., 1995; Theodoulou, 2000). As noted in Table III, SAGE detected a 5-fold increase in an ABC transporter (At3g53480) upon TNT exposure. This ABC transporter is of a class related to the Brewer's yeast (Saccharomyces cerevisiae) PDR5 gene and as such, has been suggested to have potential function as a toxin-conjugate efflux pump (Mitterbauer and Adam, 2002). Treatment of liquid-grown Arabidopsis seedlings with the nitroaromatic cytotoxin, 1-chloro-2,4-dinitrobenzene, elicited similar increases in transcript levels for three other ABC transporters, including the AtMRP1 (At1g30400) and AtMRP4 (At2g47800), that are thought to handle transport of GSH conjugates and anthocyanins into the vacuole (Tommasini et al., 1997).

In addition to undergoing oxidation reactions, the TNT taken up by plants is also subject to reduction reactions that target the aromatic nitro-groups, as evidenced by the amino, dinitro-derivatives of TNT identified in plant tissues by Bhadra et al. (1999b). SAGE suggests that such reduction reactions may be catalyzed by NADPH-dependent flavoenzymes, such as 12-oxophytodienoate reductase (At1g76680), which was induced 10-fold by TNT treatment. Similar in sequence to the yeast Old Yellow Enzyme, 12-oxophytodienoate reductase is related to the nitrate ester reductases (Schaller and Weiler, 1997), and another member of this family, pentaerythritol tetranitrate reductase, has been shown to increase TNT tolerance when expressed in transgenic tobacco (Nicotiana tabacum) plants (French et al., 1999). Thus, 12-oxophytodienoate reductase may prove a useful target for overexpression experiments aimed at increasing plant tolerance to TNT and other nitroaromatic compounds.

The observation that two tags representing the NITRILASE2 transcript (At3g44300) and a likely splice variant were strongly up-regulated by TNT exposure were initially of some concern because strong induction of this gene was previously noted during leaf senescence (Quirino et al., 1999). Other leaf senescence transcripts that were induced by TNT exposure included those for -VACUOLAR PROCESSING ENZYME (At4g32940) and GLYOXALASE II (At1g53580), which were up-regulated 7- and 3-fold, respectively (data not shown). The transcript for a cinnamyl alcohol dehydrogenase (At5g19440) was also induced 5-fold by TNT exposure, but this was not the same as either of the two CAD genes (SAG25/At4g37990 and SAG26/At1g09500) Quirino et al. (1999) found to be up-regulated in senescent leaves. No tags for either of the CAD genes studied by Quirino et al. (1999) were detected in the SAGE study, nor were tags for the transcript encoding the MtN3-like protein (SAG29/At5g13170) that they examined. Finally, SAGE detected only a 1.3-fold induction by TNT of the ribosomal protein L10 transcript (At1g14320) that was shown to be strongly up-regulated during leaf senescence. Thus, although gene expression patterns in senescing Arabidopsis roots have not been characterized to an extent that allows direct comparison of the two tissues, the SAGE data does not appear to indicate a senescence response in TNT-treated roots similar to that seen in senescent leaves.

Gene Repression in Response to TNT

Inference of metabolic function for genes whose expression is induced by stress can be relatively straightforward given sufficient information on the activity of the encoded proteins. However, understanding the down-regulation of other genes under the same conditions of stress is much more difficult because their repression may be the indirect outcome of regulatory shifts necessary to induce protective genes. Among the transcripts most repressed by TNT exposure (Table IV) were three members of the protease inhibitor/seed storage/LTP family whose products (AIR1A and AIR1B) have previously been associated with lateral root formation in response to auxin (Neuteboom et al., 1999) or response to aluminum (pEARLI-1; Richards and Gardner, 1995). These three genes are adjacent to each other on chromosome 4, but no previous evidence suggests their coordinate regulation. Another group of related genes found to be repressed were the major latex protein homologs, including a Betula vI allergen homolog. Although the function of these proteins is currently unknown, they are generally thought to accumulate in vacuoles. Thus, it is possible that their repression reflects changes in vacuole metabolism brought about by sequestration of TNT-conjugates in this organelle. Changes in vacuolar metabolism might also explain the repression of the tonoplast intrinsic protein and membrane intrinsic protein channels noted in Table IV.

This study was undertaken to improve our understanding of the mechanisms involved in plant tolerance and metabolism of xenobiotic compounds, particularly TNT. Results from these experiments suggest the involvement of previously unappreciated enzymes and, at the same time, strengthen some existing theories of how plants cope with toxic compounds. Identification of the genes involved in the metabolism of TNT should provide for focused engineering attempts to create plants better suited to remediation.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material, Growth Conditions, and Root Tissue Isolation

Arabidopsis ecotype Columbia seeds (WT-2, Lehle Seeds, Round Rock, TX) were surface-sterilized and placed in sterile Murashige and Skoog liquid medium prepared according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Plants were grown for 14 d at 25°C under a 16-h photoperiod with constant shaking at 85 rpm in a growth chamber. TNT was obtained from the U.S. Army Center for Environmental Health Research (Fort Detrick, MD). Toxicity was assessed by adding TNT from a stock solution in dimethyl sulfoxide (DMSO) to yield final concentrations of 0, 5, 10, 15, 20, 25, 30, and 40 mg L^–1 in Murashige and Skoog medium, and cultures were run in triplicate. The cultures were returned to the growth chamber for 5 d, during which time the seedlings in each flask were examined for signs of stress (leaf chlorosis and necrosis). A final concentration of 15 mg L^–1 TNT was judged to produce notable stress in the plants without causing death. Root tissues for SAGE library construction were isolated from seedlings grown 14 d in liquid Murashige and Skoog medium and dosed with TNT to a final concentration of 15 mg L^–1. Control tissues were isolated from seedlings grown under the same conditions and dosed with an equivalent volume of DMSO. The seedlings were grown in the presence of TNT or DMSO for 24 h, after which they were submerged briefly in dH₂O to remove excess medium, and excised roots were immediately frozen in liquid nitrogen. To obtain sufficient biomass, five flasks of seedlings (70–80 seeds in 200 mL of medium in a 500-mL flask) received each treatment, and treatments were replicated on three separate occasions. Root tissues were pooled by treatment and stored at –80°C before RNA extraction.

RNA Isolation and cDNA Synthesis

Total RNA from root tissues was extracted using the LiCl precipitation technique of Chang et al. (1993). Poly(A) RNA was isolated from total RNA using Dynabeads oligo-dT₍₂₅₎ magnetic beads (Dynal Biotech, Lake Success, NY) at a ratio of 0.25 mg of total RNA per 250 µL of Dynabeads and following the manufacturer's instructions. Double-stranded cDNA was synthesized from 5 µg of poly(A) RNA using the Superscript Choice cDNA synthesis kit (Invitrogen) and following the manufacturer's protocol, except for the substitution of a 5'-biotin dT₍₁₈₎ primer in the first-strand reaction.

SAGE Library Construction

SAGE libraries were constructed according to the SAGE Detailed Protocol, v1.0c (Velculescu et al., 1997), a brief description of which follows. Biotinylated cDNAs from each tissue sample were bound to streptavidincoated magnetic beads and digested with NlaIII, a restriction enzyme recognizing the four-base sequence, CATG (anchoring enzyme). DNA released by this digestion was washed away, and the beads, with the adherent 3' ends of each cDNA, were split into two pools. Linkers containing a binding site for BsmF1 (a type II restriction endonuclease—the tagging enzyme), but different sites for PCR primers were ligated to the NlaIII cleavage site at the 5' ends of the bead-bound cDNA fragments in each pool. Both pools of cDNAs were digested with BsmF1 to release SAGE tags from the beads, after which the pools were combined, and 102-bp linker-flanked ditags were formed by blunt-end ligation. Following amplification of the ditags by PCR, the linkers were removed by NlaIII digestion, and ditags were ligated to form concatemers. The concatemers were subsequently size-fractionated, ligated into the pZero vector (Invitrogen), cloned, and sequenced.

SAGE Data Analysis

Sequence files were compiled and analyzed using the SAGE Software v3.03, provided by Dr. Kenneth Kinzler (Johns Hopkins University, Baltimore). Tags containing linker sequences and repeated ditags were excluded before analysis. Because the library representing TNT-treated roots was sequenced to a slightly greater extent (32,203 tags for TNT treatment versus 31,973 tags for the control), values for the control library tags were normalized before making comparisons of relative gene expression. Ratios were used to compare the relative expression of tags between the two libraries (e.g. TNT/control), and in instances where a particular tag was absent from a library, a value of 1 was substituted to avoid division by zero. Using the SAGE software, Monte Carlo simulations were performed to estimate the statistical significance of any differential expression. The null hypothesis for these analyses was that the abundance, type, and distribution of transcripts were the same in both libraries. Assuming this null hypothesis, the reported P-chance values represent the fraction of simulations that yielded differences equal to or greater than the observed differences. This is the relative probability of obtaining the observed differences due to random variation, as previously detailed by Zhang et al. (1997).

Gene Identification

To identify the genes from which tags were derived, each 10-base tag plus the 4-base NlaIII recognition sequence was first compared against the AGI database of model genes using the Patmatch analysis tool available on The Arabidopsis Information Resource server (http://www.Arabidopsis.org). If the tag was found to match exactly the NlaIII site closest to the 3' end of a model gene, this identity was accepted for the tag. Tags that could not be found in the model gene database were compared against all Arabidopsis sequences in GenBank using the same Patmatch tool. Exact matches were annotated accordingly. All listed annotations were current as of July 31, 2003.

Quantitative PCR

Total RNA from Arabidopsis plants grown under conditions identical to those used to generate RNA for the SAGE studies was used to independently verify the expression of selected genes by quantitative RT-PCR. mRNA isolated using Dynal Oligo dT₂₅ magnetic beads served as template for single-stranded cDNA synthesis using Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Real-time fluorescent detection of RT-PCR products was performed using an ABI Prism 7700 sequence detection system and Sybr Green PCR Master Mix (Applied Biosystems). The following PCR primers were designed for this study using Primer Express v. 1.0 (Applied Biosystems): cytochrome P450 (At3g28740) forward, 5'-TTGATGCCTTTTGGGATTGG-3', and reverse, 5'-CAAGGTCACTAGCCGTTGAGC-3'; 60S ribosomal protein L23A (At2g39460) forward, 5'-TCCAGACCAAGAAAGTGAACACA-3', and reverse, 5'-CATAGTCTGGTGTAAGCCTCACGT-3'; and tonoplast intrinsic protein (At2g36830) forward, 5'-GCTTCTCGGCTCCGTCG-3', and reverse, 5'-GGCACAGCCAAGCCACC-3'. Amplification reactions were carried out according to the manufacturer's specifications as follows: 2 h at 50°C followed by a 10-min activation of the enzyme at 95°C, and 40 subsequent cycles consisting of 95°C for 15 s followed by 60°C for 1 min. All amplification reactions were run using a dilution series of cDNA from either control or TNT-treated tissues using the same PCR master mix. Amplimers were checked for purity and size by gel electrophoresis to ensure that the correct sequence was amplified. Control reactions omitting reverse transcriptase were run for all samples to ensure that genomic DNA contamination did not contribute to the amplified products. The reported values for qPCR measurements were generated from the C_t values according to the following formula:

where m and y were the slope and y intercept, respectively, for the graph of fluorescence intensity at C_t versus template quantity (log 10).

	ACKNOWLEDGMENTS

 
We thank Arthur Karnaugh for help with DNA sequencing protocols and Caroline Stevens for assistance with data analysis. Thanks also to MacArthur Long and Steve McCutcheon for help in initiating the project and Jeff Dangl for comments on the manuscript.

Received June 3, 2003; returned for revision July 14, 2003; accepted August 9, 2003.

	FOOTNOTES

 
This paper has been reviewed in accordance with the U.S. Environmental Protection Agency's peer and administrative review policies and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.028019.

¹ This work was supported by the U.S. Environmental Protection Agency (National Network for Environmental Management Studies Fellowship U–91587201–0 to D.R.E.),

^* Corresponding author; e-mail jeffdean{at}uga.edu; fax 706–583–0881.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815[CrossRef][Medline]

Beuerle T, Pichersky E (2002) Purification and characterization of benzoate: coenzyme A ligase from Clarkia breweri. Arch Biochem Biophys 400: 258–264[CrossRef][ISI][Medline]

Bhadra R, Wayment DG, Hughes JB, Shanks JV (1999a) Confirmation of conjugation processes during TNT metabolism by axenic plant roots. Environ Sci Technol 33: 446–452[CrossRef]

Bhadra R, Wayment DG, Hughes JB, Shanks JV (1999b) Characterization of oxidation products of TNT metabolism in aquatic phytoremediation systems of Myriophyllum aquaticum. Environ Sci Technol 33: 3354–3361[CrossRef]

Cataldo DA, Harvey SD, Fellows RJ, Bean RM, McVeety BD (1989) An Evaluation of the Environmental Fate and Behavior of Munitions Material (TNT, RDX) in Soil and Plant Systems (Report AD-A223, 546). Pacific Northwest Laboratories, Richland, WA

Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113–116

Davies J, Caseley JC (1999) Herbicide safeners: a review. Pestic Sci 55: 1043–1058[CrossRef]

Evans SJ, Datson NA, Kabbaj M, Thompson RC, Vreugdenhil E, De Kloet ER, Watson SJ, Akil H (2002) Evaluation of Affymetrix gene chip sensitivity in rat hippocampal tissue using SAGE analysis: Serial Analysis of Gene Expression. Eur J Neurosci 16: 409–413[CrossRef][ISI][Medline]

French CE, Rosser SJ, Davies GJ, Nicklin S, Bruce NC (1999) Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase. Nat Biotechnol 17: 491–494[CrossRef][ISI][Medline]

Hannink NK, Rosser SJ, Bruce NC (2002) Phytoremediation of explosives. Crit Rev Plant Sci 21: 511–538

Hannink N, Rosser SJ, French CE, Basran A, Murray JAH, Nicklin S, Bruce NC (2001) Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase. Nat Biotechnol 19: 1168–1172[CrossRef][ISI][Medline]

Ishikawa T (1992) The ATP-dependent glutathione S-conjugate export pump. Trends Biochem Sci 17: 433–438

Ishikawa T, Li Z-S, Lu Y-P, Rea PA (1997) The GS-X pump in plant, yeast, and animal cells: structure, function, and gene expression. Biosci Rep 17: 189–207[CrossRef][ISI][Medline]

Jones SJ, Riddle DL, Pouzyrev AT, Velculescu VE, Hillier L, Eddy SR, Stricklin SL, Baillie DL, Waterston R, Marra MA (2001) Changes in gene expression associated with developmental arrest and longevity in Caenorhabditis elegans. Genome Res 11: 1346–1352[Abstract/Free Full Text]

Kreuz K, Gaudin J, Stingelin J, Ebert E (1991) Metabolism of aryloxyphenoxypropanoate herbicide, CGA 184927, in wheat, barley and maize: differential effects of the safener CGA 185072. Z Naturforsch 46: 901–905

Kreuz K, Tommasini R, Martinoia E (1996) Old enzymes for a new job: herbicide detoxification in plants. Plant Physiol 111: 349–353[ISI][Medline]

Li Z-S, Zhao Y, Rea PA (1995) Magnesium adenosine 5'-triphosphateenergized transport of glutathione-S-conjugates by plant vacuolar membrane vesicles. Plant Physiol 107: 1257–1268[Abstract]

Lorenz WW, Dean JFD (2002) SAGE profiling and demonstration of differential gene expression along the axial developmental gradient of lignifying xylem in loblolly pine (Pinus taeda). Tree Physiol 22: 301–310[ISI][Medline]

Lucero ME, Mueller W, Hubstenberger J, Phillips GC, O'Connell MA (1999) Tolerance to nitrogenous explosives and metabolism of TNT by cell suspensions of Datura innoxia. In Vitro Cell Dev Plant 35: 480–486

MacIntosh GC, Wilkerson C, Green PJ (2001) Identification and analysis of Arabidopsis expressed sequence tags characteristic of non-coding RNAs. Plant Physiol 127: 765–776[Abstract/Free Full Text]

Madden S, Galella E, Zhu J, Bertelsen A, Beaudry G (1997) SAGE transcript profiles for p53-dependent growth regulation. Oncogene 15: 1079–1085[CrossRef][ISI][Medline]

Martinoia E, Grill E, Tommasini R, Kreuz K, Amrhein M (1993) ATP-dependent glutathione S-conjugate export pump in the vacuolar membrane of plants. Nature 364: 247–249[CrossRef]

Matsumura H, Nirasawa S, Terauchi R (1999) Technical advance: transcript profiling in rice (Oryza sativa L.) seedlings using serial analysis of gene expression. Plant J 20: 719–726[CrossRef][ISI][Medline]

Mitterbauer R, Adam G (2002) Saccharomyces cerevisiae and Arabidopsis thaliana: useful model systems for the identification of molecular mechanisms involved in resistance of plants to toxins. Eur J Plant Pathol 108: 699–703[CrossRef]

Neuteboom LW, Ng JMY, Kuyper M, Clijdesdale OR, Hooykaas PJJ, van der Zaal BJ (1999) Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation. Plant Mol Biol 39: 273–287[CrossRef][ISI][Medline]

Paquette SM, Bak S, Feyereisen R (2000) Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana. DNA Cell Biol 19: 307–317[CrossRef][ISI][Medline]

Patankar S, Munasinghe A, Shoaibi A, Cummings LM, Wirth DF (2001) Serial analysis of gene expression in Plasmodium falciparum reveals the global expression profile of erythrocytic stages and the presence of anti-sense transcripts in the malarial parasite. Mol Biol Cell 12: 3114–3125[Abstract/Free Full Text]

Pavlostathis SG, Jackson GH (1999) Biotransformation of 2,4,6-trinitrotoluene in Anabaena sp. cultures. Environ Toxicol Chem 18: 412–419

Quirino BF, Normanly J, Amasino RM (1999) Diverse range of gene activity during Arabidopsis thaliana leaf senescence includes pathogen-independent induction of defense-related genes. Plant Mol Biol 40: 267–278[CrossRef][ISI][Medline]

Richards KD, Gardner RC (1995) pEARLI 1 (accession no. L43080): an Arabidopsis member of a conserved gene family (PGR95–099). Plant Physiol 109: 1497[CrossRef][Medline]

Riggins GJ, Strausberg RL (2001) Genome and genetic resources from the Cancer Genome Anatomy Project. Hum Mol Genet 10: 663–667[Abstract/Free Full Text]

Schaller F, Weiler EW (1997) Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana: structural and functional relationship to yeast old yellow enzyme. J Biol Chem 272: 28066–28072[Abstract/Free Full Text]

Schröder P, Collins C (2002) Conjugating enzymes involved in xenobiotic metabolism of organic xenobiotics in plants. Intl J Phytoremed 4: 247–265

Schultz TF, Kiyosue T, Yanovsky M, Wada M, Kay SA (2001) A role for LKP2 in the circadian clock of Arabidopsis. Plant Cell 13: 2659–2670[Abstract/Free Full Text]

Stöllberg J, Urschitz J, Urban Z, Boyd CD (2000) A quantitative evaluation of SAGE. Genome Res 10: 1241–1248[Abstract/Free Full Text]

Stromvik MV, Sundararaman VP, Vodkin LO (1999) A novel promoter from soybean that is active in a complex developmental pattern with and without its proximal 650 base pairs. Plant Mol Biol 41: 217–231[Medline]

Terryn N, Rouzé P (2000) The sense of naturally transcribed antisense RNAs in plants. Trends Plant Sci 5: 394–396[CrossRef][ISI][Medline]

Theodoulou FL (2000) Plant ABC transporters. Biochim Biophys Acta 1465: 79–103[Medline]

Tommasini R, Vogt E, Schmid J, Fromentau M, Amrhein N, Martinoia E (1997) Differential expression of genes coding for ABC transporters after treatment of Arabidopsis thaliana with xenobiotics. FEBS Lett 411: 206–210[CrossRef][Medline]

Vaistij FE, Jones L, Baulcombe DC (2002) Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14: 857–867[Abstract/Free Full Text]

Velculescu VE, Madden SL, Zhang L, Lash AE, Yu J, Rago C, Lal A, Wang CJ, Beaudry GA, Ciriello KM (1999) Analysis of human transcriptomes. Nat Genet 23: 387–388[ISI][Medline]

Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene expression. Science 270: 484–487[Abstract/Free Full Text]

Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai MA, Bassett DE, Hieter P, Vogelstein B, Kinzler KW (1997) Characterization of the yeast transcriptome. Cell 88: 243–251[CrossRef][ISI][Medline]

Walsh ME, Jenkins TF, Schnitcker PS, Elwell JW, Stutz MH (1993) USA Cold Regions Research and Engineering Laboratory CRREL Special Report 93–5. USA Cold Regions Research and Engineering Laboratory, Hanover, NH, pp 1–17

Yang O, Reinhard K, Schiltz E, Matern U (1997) Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L. Plant Mol Biol 35: 777–789[CrossRef][ISI][Medline]

Zhang L, Zhou W, Velculescu VE, Kern SE, Hruban RH, Hamilton SR, Vogelstein B, Kinzler KW (1997) Gene expression profiles in normal and cancer cells. Science 276: 1268–1272[Abstract/Free Full Text]

Related articles in Plant Physiol.:

Peter V. Minorsky
Plant Physiol. 2003 133: 1038-1039. [Full Text]  

This article has been cited by other articles:

				V. Srinivasasainagendra, G. P. Page, T. Mehta, I. Coulibaly, and A. E. Loraine CressExpress: A Tool For Large-Scale Mining of Expression Data from Arabidopsis Plant Physiology, July 1, 2008; 147(3): 1004 - 1016. [Abstract] [Full Text] [PDF]

				F. Alferez, G. Y. Zhong, and J. K. Burns A citrus abscission agent induces anoxia- and senescence-related gene expression in Arabidopsis J. Exp. Bot., July 1, 2007; 58(10): 2451 - 2462. [Abstract] [Full Text] [PDF]

				J. Bao, S. Lee, C. Chen, X. Zhang, Y. Zhang, S. Liu, T. Clark, J. Wang, M. Cao, H. Yang, et al. Serial Analysis of Gene Expression Study of a Hybrid Rice Strain (LYP9) and Its Parental Cultivars Plant Physiology, July 1, 2005; 138(3): 1216 - 1231. [Abstract] [Full Text] [PDF]

				V. Poroyko, L.G. Hejlek, W.G. Spollen, G.K. Springer, H.T. Nguyen, R.E. Sharp, and H.J. Bohnert The Maize Root Transcriptome by Serial Analysis of Gene Expression Plant Physiology, July 1, 2005; 138(3): 1700 - 1710. [Abstract] [Full Text] [PDF]

				M. P. Mezzari, K. Walters, M. Jelinkova, M.-C. Shih, C. L. Just, and J. L. Schnoor Gene Expression and Microscopic Analysis of Arabidopsis Exposed to Chloroacetanilide Herbicides and Explosive Compounds. A Phytoremediation Approach Plant Physiology, June 1, 2005; 138(2): 858 - 869. [Abstract] [Full Text] [PDF]

				R. E. Sharp, V. Poroyko, L. G. Hejlek, W. G. Spollen, G. K. Springer, H. J. Bohnert, and H. T. Nguyen Root growth maintenance during water deficits: physiology to functional genomics J. Exp. Bot., November 1, 2004; 55(407): 2343 - 2351. [Abstract] [Full Text] [PDF]

				S. J. Robinson, D. J. Cram, C. T. Lewis, and I. A.P. Parkin Maximizing the Efficacy of SAGE Analysis Identifies Novel Transcripts in Arabidopsis Plant Physiology, October 1, 2004; 136(2): 3223 - 3233. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 133/3/1397    most recent pp.103.028019v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (27) Citing Articles via Google Scholar Google Scholar Articles by Ekman, D. R. Articles by Dean, J. F.D. Search for Related Content PubMed PubMed Citation Articles by Ekman, D. R. Articles by Dean, J. F.D. Agricola