Involvement of Ethylene in Stress-Induced ... - Plant Physiology

Involvement of Ethylene in Stress-Induced Expression of the TLC1.1 Retrotransposon from Lycopersicon chilense Dun.1[w] Gerardo Tapia, Isabel Verdugo, Mońica Yan˜ez, Ivań Ahumada, Cristina Theoduloz, Cecilia Cordero, Fernando Poblete, Enrique Gonza´lez, and Simoń Ruiz-Lara* Instituto de Biologıá Vegetal y Biotecnologıá, Universidad de Talca, Talca, Chile

The TLC1 family is one of the four families of long terminal repeat (LTR) retrotransposons identified in the genome of Lycopersicon chilense. Here, we show that this family of retroelements is transcriptionally active and its expression is induced in response to diverse stress conditions such as wounding, protoplast preparation, and high salt concentrations. Several stressassociated signaling molecules, including ethylene, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid, are capable of inducing TLC1 family expression in vivo. A representative of this family, named TLC1.1, was isolated from a genomic library from L. chilense. Transient expression assays in leaf protoplasts and stably transformed tobacco (Nicotiana tabacum) plants demonstrate that the U3 domain of the 5#-LTR region of this element can drive stress-induced transcriptional activation of the b-glucuronidase reporter gene. Two 57-bp tandem repeated sequences are found in this region, including an 8-bp motif, ATTTCAAA, previously identified as an ethylene-responsive element box in the promoter region of ethyleneinduced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the b-glucuronidase gene shows that these elements are required for ethylene-responsive gene expression in protoplasts and transgenic plants. We suggest that ethylene-dependent signaling is the main signaling pathway involved in the regulation of the expression of the TLC1.1 element from L. chilense.

Retrotransposons are mobile genetic elements that share several structural and functional characteristics with retroviruses (Boeke and Corces, 1989). They are widely distributed throughout all eukaryotic genomes examined so far (Xiong and Eickbush, 1990) and constitute the major class of mobile genetic elements found in plants (Flavell et al., 1992; Grandbastien, 1992; Voytas et al., 1992). Two classes of retrotransposons have been identified: the long terminal repeat (LTR) retrotransposons (flanked by LTRs) and the nonLTR retrotransposons. Regarding their sequence homology and gene organization, two major groups of LTR retroelements, the Ty1/copia and the Ty3/gypsy, have been distinguished (Grandbastien, 1992); representatives of both groups have been found in higher plants (Sentry and Smyth, 1989; Smyth et al., 1989; Flavell et al., 1992; Voytas et al., 1992). Even though most of the identified plant LTR retrotransposons appear to be inactive, evidence for element mobility is available for the maize (Zea mays) Bs1 and Zeon-1 elements (Johns et al., 1985; Hu et al., 1995), the tobacco (Nicotiana tabacum) Tnt1 and Tto1 elements (Grandbastien et al., 1989; Hirochika, 1993), the Tnp2 1

This work was supported by the Fondo Nacional de Ciencia y Tecnologıá (grant no. 1980387) and the Programa de Investigacioń en Biotecnologıá Vegetal, Direccioń de Investigacioń y Asistencia Tećnica, from Universidad de Talca. * Corresponding author; e-mail [email protected]; fax 56–71–200268. [w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.059766.

element of Nicotiana plumbaginifolia (Vaucheret et al., 1992), the Tos elements of rice (Oryza sativa; Hirochika et al., 1996), and the BARE-1 retrotransposon of barley (Hordeum vulgare; Suoniemi et al., 1996a, 1996b). The retrotransposition process includes transcription and reverse transcription (RT) steps, which are modulated by the host, the retroelement itself, or by external signals (for review, see Kumar and Bennetzen, 1999). Transcription is a key event in duplicative transposition and is involved in both the production of genomic RNA used as template for RT and the production of mRNA species required for the synthesis of proteins necessary for transposition (Grandbastien et al., 1994). In plant retrotransposons, the transcription step appears to be a tissue-specific and a developmentally regulated process (Pouteau et al., 1991; Grandbastien et al., 1994; Kumar and Bennetzen, 1999). Expression of the Tnt1 and Tto1 elements of tobacco (Pouteau et al., 1991, 1994; Hirochika, 1993; Moreau-Mhiri et al., 1996; Grandbastien et al., 1997) and the Tos elements of rice (Hirochika et al., 1996) was shown to be greatly increased by stress conditions, including protoplast isolation, cell culture, or pathogen attack (for review, see Wessler, 1996; Kumar and Bennetzen, 1999). Signals controlling retrotransposon expression of these elements are located within the 5# LTR and the adjacent untranslated region. Several cis-acting elements corresponding to tandem repeated sequences and specific DNA motifs have been identified in the LTR U3 domains and have been shown to be involved in the induction of retrotransposon transcription by biotic and abiotic stress factors (Casacuberta and Grandbastien,

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1993; Grandbastien et al., 1997; Vernhettes et al., 1997; Grandbastien, 1998; Takeda et al., 1998, 1999). Within this region, high variability is indeed observed among the different subfamilies of plant retrotransposons, a trait that has been recently associated with the ability of these promoters to respond to different stressassociated signaling molecules (Marillonnet and Wessler, 1998; Vernhettes et al., 1998; Araujo et al., 2001). Such regulated expression results in a tight control of transcription of these highly repetitive retroelements, and thereby may have evolved as an adaptative mechanism that allowed them to coexist with the host genome (Takeda et al., 1999; Beguiristain et al., 2001). We have identified four Ty1/copia-like retrotransposon families (TLC1, TLC2, TLC3, and TLC4) in the genome of Lycopersicon chilense and showed that these elements are transcriptionally active (Yan˜ez et al., 1998). Due to the geographic distribution of L. chilense (3,000 m above sea level in the Atacama Desert), this plant species has adapted to adverse conditions such as extreme temperatures and salt and drought stresses. Similar conditions have been suggested to affect transposable element mobility (Kalendar et al., 2000). Therefore, L. chilense represents an interesting system to analyze the molecular structure of its LTR retrotransposons, as well as the conditions that control their transcription and mobility. Here, we report the isolation and molecular characterization of TLC1.1, a representative of the TLC1 retrotransposon family from L. chilense. We show that several stress challenges as well as different stress-associated signaling molecules are able to induce in vivo expression of the TLC1.1 population. Ethylene-dependent and ethylene-independent signal transduction pathways were found to modu-

late TLC1.1 transcription. Here, we demonstrate that ethylene-dependent induction is mediated through putative ethylene-responsive element (ERE) box regulatory elements present in the U3 region of TLC1.1. RESULTS Isolation and Structural Characterization of the Retrotransposon TLC1.1

The haploid genome of L. chilense contains approximately 900 copies of the TLC1 retrotransposon family, which constitutes the most abundantly represented family of LTR retrotransposons in this genome (Yan˜ez et al., 1998). Complete copies of the TLC1 retrotransposon were obtained by screening a genomic library from L. chilense with a DNA probe corresponding to a conserved domain in the reverse transcriptase coding region within the TLC1 retrotransposon family (Yan˜ez et al., 1998). Positive clones were isolated and the presence of the entire retrotransposon in recombinant phages was analyzed by Southern-blot hybridization against probes designed to detect either the 5# end (U5PBS) or the 3# end (PPT-U3) of the retroelement. A clone containing a 6-kb insert (hereafter TLC1.1) that hybridizes with both probes was selected for further analysis. The 5,248-bp complete nucleotide sequence of TLC1.1 (GenBank accession no. AF279585) showed that, as reported for other LTR retrotransposons, this retrolement is composed of a single open reading frame (ORF; 3,987 bp) coding for the gag and pol (prot, endo, and RT) domains flanked by two LTRs. DNA sequence analysis of the LTR regions showed a 5# LTR of 578 bp and a 3# LTR of 562 bp (Fig. 1). These two

Figure 1. Deduced structure of the L. chilense TLC1.1 retrotransposon. The 5#-LTR region is shown amplified. The regions hybridizing to U5-PBS and PPT-U3 are marked, and arrows indicate the positions of the amplification primers. 2076

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Involvement of Ethylene in Stress-Induced Expression of TLC1.1

regions share 85% identity, which suggests that TLC1.1 corresponds to an ancient insertion (San Miguel et al., 1998). The sequences of the different domains of TLC1.1 were compared with those reported for other plant retrotransposons using the BioEdit software package (Hall, 1999) and results from such comparisons are summarized in Table I. Homology analysis of the ORF encoding the TLC1.1 polyprotein showed the highest degree of similarity with Retrolyc1-1 (partial sequence) from the related species Lycopersicon peruvianum (Costa et al., 1999) and with Tnt1 from tobacco (94% and 87%, respectively). However, when the analysis was restricted to the LTR region and its domains, a lower degree of similarity was observed between TLC.1.1 and Tnt1, albeit the homology with Retrolyc1-1 was conserved. Expression Analysis of the Retrotransposon TLC1

We have previously reported that various TLC retrotransposon families from L. chilense are expressed in leaf protoplasts (Yan˜ez et al., 1998). To get additional insight into this pattern of expression, transcriptional activity of this element was investigated in different tissues from mature plants. Total RNA was purified from roots, leaves, and inflorescences of plants and the transcription of TLC1 was determined by RT-PCR analysis. As control, RT-PCR reactions designed to amplify a 450-bp region of the 18S rRNA were performed. As shown in Figure 2, the amplification product corresponding to the expected size of TLC1.1 PPT-U3 cDNA was detected only in RNA samples obtained from roots (Fig. 2, lane 4). No amplification of TLC1 was observed in samples from leaves or inflorescences (Fig. 2, lanes 2 and 3), while 18S was present in all RNA samples (Fig. 2, lanes 5–7). Cloning of the amplification product obtained from roots and analysis of the nucleotide sequences of 10 randomly selected clones indicate that a population of different TLC1 representative elements was expressed in this tissue (data not shown), ruling out the possibility that this product might be originated by read-through transcription of a specific TLC1 copy by a promoter of a flanking gene. Taken together, these results indicate Table I. Nucleotide sequence comparison of TLC1.1 retrotransposon with other similar retroelements The ORF, 5#-LTR complete region, and the U3 and U5 domains of TLC1.1 (AF279585) were compared with the respective regions of the tobacco retrotransposon Tnt1-94 (X13777), the tobacco retrotransposon Tto1 sequence encoding an ORF (D83003), and the L. peruvianum retrotransposon Retrolyc1-1 partial sequence (AF228701). Identity was scored using BioEdit software package (Hall, 1999). Tnt1 Tto1 Retrolyc a

ORFa

ORFb

5# LTR

U3

U5

87% 65.3% 94%

84.8% 48.4% N/Ac

63.2% 54.1% 86.7%

55.1% 56% 92%

69.4% 52.7% 83.2%

Nucleotide homology. protein sequence described.

b

Amino acid homology.


c

N/A, No

Figure 2. RT-PCR analysis of tissue-specific in vivo transcription of TLC1 retrotransposon in L. chilense plants. Lanes 2 to 4, Detection of TLC1 mRNA by amplification reactions using primer P1 and P2. Lanes 5 to 7, Amplification of 18S rRNA using primers P181 and P182 (control reactions). RNA samples were extracted from leaves (lanes 2 and 5), inflorescences (lanes 3 and 6), and roots (lanes 4 and 7). Lane 1, Control PCR with a pTLC1.1, a pUC19 derivative clone containing TLC1.1 retrotransposon. Lanes 8 and 9, Control PCR without RT performed with root RNA using P1-P2 and P181-P182 primers, respectively. M, 100-bp Mr marker (Invitrogen).

that TLC1 expression is restricted to root tissues in nonstressed plants. The induction of the transcription of TLC1 retrotransposon in leaves of L. chilense plants exposed to different stress conditions was also investigated (i.e. exposure to cell wall-degrading enzymes, mechanical wounding, drought, and salt stress). Total RNA isolated at different times of stress exposure and the U5-PBS probe (see Fig. 1) were used to detect TLC1.1 transcription by northern-blot hybridization. While transcription levels of the large subunit Rubisco gene remained unchanged in both stressed and control plants, a clear induction of TLC1.1 transcription was observed in challenged plants (Fig. 3). Two transcripts of approximately 6 and 5.3 kb were detected as early as 2 h after mechanical wounding, with a maximal expression at 12 h of induction (Fig. 3). Similar results were also obtained when cell wall-degrading cellulases were used as the stress challenge (data not shown). Whereas the 5.3-kb RNA would correspond to the expected size of a full-length TLC1.1 transcript, the nature of the 6-kb RNA is unclear. Nevertheless, longer hybridizing transcripts have also been reported in RNA-blot analysis with the Tnt1 retrotransposon probe (Casacuberta and Grandbastien, 1993), indicating that this longer transcript might correspond to a TLC1.1-derived RNA. The effect of drought and salt stresses on TLC1.1 expression was analyzed by RT-PCR and compared with the expression of the L. chilense endochitinase and H1 histone-like genes, both reported to be induced by drought (Chen et al., 1994; Wei and O’Connell, 1996). As shown in Figure 4, an amplified product corresponding to the expected size of the TLC1.1 U3 domain was obtained only in plants under high salt stress, without any detectable expression in drought-stressed plants for up to 10 d. However, activation of the endochitinase and H1 histone-like genes was detected under drought and salt stress conditions in control experiments. Amplified 2077

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Figure 3. Northern analysis of TLC1.1 transcription in leaves from L. chilense plants exposed to wounding stress. W, Mechanically wounded plants hybridized with the U5-PBS probe; R, hybridization control using a probe for the large subunit of Rubisco mRNA. The size of the hybridizing mRNA is indicated.

(2,4-D), abscisic acid (ABA), and H2O2 on in vivo retroelement expression. Total RNA was isolated from L. chilense leaves after 24 h of treatment and hybridized to the U5-PBS probe (Fig. 1). Figure 5 shows that a band corresponding to the expected size of TLC1.1 was obtained in plants treated with ethylene, methyl jasmonate, salicylic acid, and 2,4-D, but not in plants treated with ABA or H2O2. These results suggest the presence of specific sequence motifs acting as regulatory elements that modulate the transcriptional activation of the TLC1 promoters in response to different signal transduction pathways. Functional Analysis of the 5#-LTR Region of TLC1.1

TLC1 products were cloned, and 10 randomly selected clones were characterized. Sequence analysis indicated that five clones were identical to TLC1.1, while the other clones were 96% to 98% identical to this retroelement. This result shows that different members of TLC1 retrotransposon family are induced in response to salt stress, discarding the possibility that the transcripts were produced by read-through transcription of a specific TLC1 copy by a salt-inducible promoter of a flanking gene. Taken together, these results indicate that expression of TLC1 is not induced by every environmental challenge to which L. chilense is exposed in its natural habitat and suggest that signaling pathways other than those triggered by osmotic stress may be involved in regulating TLC1 expression in response to high salt. Differential Induction of TLC1 Transcription by Stress-Related Signaling Molecules

To get an insight into signal transduction pathways controlling the activation of the TLC1 retrotransposon, we investigated the effect of ethylene, methyl jasmonate, salicylic acid, 2,4-dichlorophenoxyacetic acid

To study the role of the 5#-LTR domain in controlling the transcriptional activity of TLC1.1, a more detailed analysis of this region was performed. The nucleotide sequence of the 5#-LTR region was analyzed to identify potential cis-regulatory elements involved in the response to the above signal molecules. Search for responsive element motifs was performed in silico by means of the Promoter Scan Program (Prestridge, 2000). Based on nucleotide sequence comparisons of the 5# LTR with the respective regions in the Tnt1 retrotransposon from tobacco and Retrolyc1-1 (partial sequence) from L. peruvianum, three domains have been defined: the U3 domain (approximately from nucleotides 1–226), the R domain (nucleotides 227– 233), and the U5 domain (nucleotides 234–578). A particular array of putative transcriptional regulatory elements was identified in the U3 domain of the 5# LTR. As shown in Figure 6, this region includes a near consensus TATA-box between nucleotides 194 and 200 and, notoriously, two 57-bp tandem repeated sequences (TRS1 and TRS2), sharing a 95% identity, in positions 38 to 94 (TRS1) and 95 to 151 (TRS2), respectively. A sequence motif that matches with the socalled ERE box (Itzhaki et al., 1994) was found within

Figure 4. Analysis of TLC1.1 transcription under salt and drought stress. RT-PCR analysis was performed using total RNA obtained from stressed plant leaves (1–12). TLC1.1 transcription was determined through amplification reactions with specific primers L00 and P2 (lanes 1, 4, 7, and 10). As a control, the expression of the osmotic stress-induced tomato genes coding for endochitinase protein (lanes 2, 5, 8, and 11) and H1 histone-like protein (lanes 3, 6, 9, and 12) is also included, using the endo1and endo2- or his1- and his2-specific primers, respectively. P, Control PCR with genomic DNA of L. chilense with L00 and P2 primers; R and C, RT-PCR with total RNA from nonstressed leaves with large subunit Rubisco and TLC1.1 specific primers, respectively. M, 100-bp Mr standard (Promega). 2078



Figure 5. Expression of TLC1.1 induced by different signaling molecules. Hybridization was performed using total RNA obtained from leaves of L. chilense at 24 h after treatment with the indicated compounds. 10 mg of total RNA per lane were separated on a 1.0% formaldehyde-agarose gel and transferred to a nylon membrane. The blot was hybridized with a-32P-dCTP random prime-labeled U5-PBS fragment. Equal loading of RNA was confirmed by ethidium bromide staining of the rRNA. Treatments were as described in ‘‘Materials and Methods.’’ Concentrations used were 1 mM salicylic acid, 50 mM methyl jasmonate, 100 mM ABA, 50 mM 2,4-D, or 1% v/v H2O2. Control plants were sprayed with water/0.01% ethanol/0.01% methanol.

both TRS1 and TRS2 sequences. No other canonical sequences corresponding to known cis-elements were found in either the U3 or the U5 domains. To establish the role of the above-mentioned elements in LTR promoter activity, a 578-bp DNA fragment, containing the complete LTR region (P578) or a 270-bp fragment corresponding to the U3 domain located at the 5#-end of the LTR region (P270), was fused to the b-glucuronidase (GUS) reporter gene to generate the constructs pTG578 and pTG270, respectively (Fig. 7A). These plasmids were electroporated into leaf protoplasts, and GUS activity was analyzed in transient expression assays after a 12-h incubation period. The results of such experiments are shown in Figure 7B. Both fragments, P578 and P270, were able to direct GUS expression with higher levels than those observed when the reporter gene was fused to the cauliflower mosaic virus (CaMV) 35S promoter (4-fold and 2-fold, respectively). Although GUS activity observed with the P270 construct was almost one-half of that detected with the complete 5# LTR, the U3-R domain appeared to be sufficient to promote high GUS expression, indicating that transcriptional regulatory sequences are contained within this domain. In agreement with this observation, only background levels of GUS activity were observed in protoplasts transformed with a construct containing the LTR U5 region fused to GUS (data not shown).

in GUS expression (about 50%) was observed with both single and double mutants (Fig. 7C). To confirm that the ERE boxes in the U3 domain are involved in the response to ethylene, we assayed the ability of pTG270 and single and double mutant constructs to direct GUS expression in response to exogenously applied ethylene. L. chilense protoplasts were electroporated with the respective constructs and divided into two fractions that were treated with air or 10 mL L21 ethylene for 12 h before determining GUS activity. A 3-fold increase in GUS activity was observed only in ethylene-treated protoplasts transformed with the pTG270 construct, and an insignificant increase in GUS activity was observed when protoplasts were transformed with either single or double mutant constructs or when the GUS gene was fused to the CaMV 35S promoter (Fig. 7D). These results indicate that the P270 promoter is able to respond to ethylene and that this response requires both ERE boxes for full promoter activity. Since mutation of both ERE boxes did not abolish completely the ability of P270 to activate GUS expression, another yet unidentified cis-element may also be present in the U3 domain of TLC1.1. Function of the ERE boxes in mediating P270 response to ethylene was further confirmed in transgenic tobacco plants. Fragments P270 and PTRS1*/2* were fused to the GUS reporter gene in the plasmid pBI121 to generate the plasmids pBI270 and pBI1*/2*, respectively. These constructs were introduced by electroporation into the Agrobacterium tumefaciens strain LBA4404 and used to transform tobacco var Xanthi plants by the leaf disc infection method (Horsch et al., 1984). After transforming with pBI270 and pBI1*/2*, 35 and 24 transgenic lines were obtained, respectively, and the presence of the inserts was confirmed by PCR analysis. The insertion copy number for these constructs was determined by Southern analysis over HindIII- or EcoRI-digested genomic DNA using a 620-bp fragment corresponding to either

The ERE Motifs Mediate Ethylene-Induced Activation of the U3 Promoter Region

To analyze the role of the putative ERE boxes in controlling TLC1.1 expression, the ERE box sequence motifs contained in the TRS1 and TRS2 repeats within the U3 domain were modified by in vitro mutagenesis of the P270 promoter fragment. The ERE box motif ATTTCAAA was modified to CTGCAGAA in TRS1, and to TCTAGAAG in TRS2. Single (pTRS1* and pTRS2*) and double (pTRS1*/2*) mutant constructs were then generated by fusing these fragments to the GUS gene (Fig. 7A) and introduced into L. chilense leaf protoplasts by electroporation. A significant decrease Plant Physiol. Vol. 138, 2005

Figure 6. Partial nucleotide sequence of TLC 1.1 5#-LTR region showing the first 270 nucleotides of TLC 1.1 5#-LTR region comprising the U3 domain (nt 1–226), the R domain (nt 227–232), and part of the U5 domain (nt 233–270). The 57-bp tandem repeated sequences TRS1 and TRS2 are underlined, and the ERE box motifs are in bold and indicated by double arrows. The putative TATA promoter element is boxed. 2079

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Figure 7. Transient expression analysis in L. chilense leaf protoplasts. Protoplasts were trasnsformed with 10 mg of various constructs containing the complete LTR or its derivatives fused to a promoterless GUS gene. A, Schematic drawing of the different constructs used in this work. B to D, GUS activity determined in protoplasts electroporated with the constructs containing either (B) the LTR region or the isolated U3 domain fused to GUS gene, (C) the U3 domain and mutant for the tandem repeated sequences TRS1 and TRS2, or (D) the U3 domain, double mutant (TRS1*/2*), and the single mutants (TRS1* and TRS2*) in the absence (black) or presence (hatched) of exogenous ethylene. Bars represent the SD from four independent experiments.

the P270-GUS or PTRS1*/2* -GUS junctions as molecular probe. Transgenic lines showing only one hybridization band were considered to contain a single insertion copy of T-DNA. Four transformants harboring a unique copy of either construct were selected for GUS expression studies. GUS activity was measured by a fluorometric assay in transgenic plants after ethylene treatment. An increase of approximately 3 times the GUS activity was observed in P270::GUS transgenic plants after 18 h incubation with the hormone (Fig. 8A). A similar increase of GUS activity was observed when the role of the ERE boxes was evaluated in response to mechanical stress (i.e. wounding; Fig. 8B). Plants containing the construct PTRS1*/ 2*::GUS did not show significant increase in GUS expression in response to ethylene treatment in either intact or damaged leaves (Fig. 8; supplemental data). To corroborate that the ERE boxes are also involved in the transcriptional activation of TLC1.1 by mechanical wounding, a histochemical analysis was performed. Leaves from transgenic plants harboring either the P270::GUS or PTRS1*/2*::GUS constructs showed GUS staining, but the intensity was much lower in those plants that contained the double ERE box mutant (Fig. 9). Taken together, these results show that the ERE boxes are functional cis-elements essen2080

tial for ethylene-induced expression of GUS gene in transgenic plants and also participate in the response to wounding stress. Our data suggest that the ERE boxes may have the same role in the transcriptional activation of TLC1.1 retrotransposon in L. chilense.

DISCUSSION Structure and Organization of TLC1.1

Retrotransposons are major constituents of higherplant genomes and provide much of the raw material for the evolution of genome structure and function. In all plant species studied so far, most elements appear to be less than 10 million years old (SanMiguel et al., 1998). Recent studies suggest that, due to a combination of mutations and epigenetic silencing, most retrotransposons are currently inactive (Wright and Voytas, 1998; Hirochika et al., 2000; Devos et al., 2002). In tomato, for instance, more than 9,000 copies of LTR retrotransposons have been described (Su and Brown, 1997; Budiman et al., 2000; Rogers and Pauls, 2000). However, to date only partial sequences of some of these retroelements have been described (Costa et al., 1999). In this article, we reported the entire sequence of Plant Physiol. Vol. 138, 2005


Figure 8. GUS expression in P270::GUS or PTRS1*/2*::GUS transgenic tobacco plants in response to exogenous ethylene or wounding. Leaves from transgenic tobacco plants harboring single copy of P270::GUS or PTRS1*/2*::GUS constructs were collected after an 18-h treatment with either 10 mL L21 ethylene or air. Collected leaves were processed for fluorometric detection of GUS activity. Four transgenic lines were analyzed and four GUS activity measurements were performed for each transformed plant. Different letters above bars indicate that differences between values are significant at P , 0.05 (Tukey’s HDS multiple comparison test following a three-way ANOVA). Bars represent mean 6 SE.

a typical copia-like retrotransposon of tomato, TLC1.1, a representative of the TLC1 family of LTR retrotransposons from the wild-type tomato L. chilense (Yan˜ez et al., 1998). The complete nucleotide sequence shows that all typical structural features of Ty1/copia class retrotransposons are found in TLC1.1. The internal region of TLC1.1 contains one large ORF including gag and pol regions, indicating that TLC1.1 synthesizes a polyprotein (Fig. 1). The conservation of essential domains for RNA binding, protease, endonuclease, reverse transcriptase, and RNAseH activities suggests that this polyprotein may be functional. The deduced PBS sequence suggests that tRNAiMet is used as a primer for first-strand synthesis by reverse transcriptase, a prevalent feature among plant retrotransposons (for review, see Kumar and Bennetzen, 1999; Casacuberta and Santiago, 2003). In addition, we showed that the TLC1.1 retrotransposon has the regulatory sequences needed to drive its transcription, which is the major regulatory step for transposition.

transcription of this retroelement is restricted to root tissues without any detectable expression in leaves or inflorescences, suggesting a tissue-specific control mechanism (Fig. 2). This appears to be a general feature in retroelement expression since root-specific expression has also been described for the Tnt1 retrotransposon (Pouteau et al., 1991; Grandbastien et al., 1994), while others such as BARE-1 from barley and PREM-2 from maize are specifically transcribed in leaves and microspores, respectively (Suoniemi et al., 1996a, 1996b; Turcich et al., 1996). Here, we show that transcription of TLC1 was induced when L. chilense plants were exposed to stress conditions. In these experiments, the transcription of TLC1 was activated in response to wounding and Onozuka R10 cellulase in a similar way to that described for other active retrotransposons (Pouteau et al., 1991; Mhiri et al., 1997; Takeda et al., 1998, 1999). These results correlate with the induction of TLC1 in response to ethylene, methyl jasmonate, and salicylic acid, which were described to be involved in signal transduction pathways associated to the abovementioned stress conditions (O’Donnell et al., 1996, 2003). When we analyzed the transcriptional activity of TLC1 in response to prevalent environmental challenges in the L. chilense habitat, a rather unexpected result was obtained. Only salt stress was able to activate transcription of the retrotransposon, with no detectable expression observed under drought stress (Fig. 4). Since most of genes involved in the response to osmotic stress are also involved in the regulation of hydric balance, they are usually induced by both high salt concentration and dehydration (Shinozaki and Yamaguchi-Shinozaki, 2000; Zhu, 2000; Seki et al., 2002). Nevertheless, besides osmotic stress, high salt concentration causes an ionic toxic effect. Several genes involved in salt tolerance are only induced by high salinity (Zhu, 2000; Seki et al., 2002). It is plausible that induction of TLC1 occurs in response to an ionic toxic effect rather than to osmotic stress.

Transcriptional Induction of TLC1 in Response to Stress Conditions

The genomic shock model proposed by McClintock (1984) postulates that mobile genetic elements play a crucial role in genome restructuring induced by environmental challenges. According to this theory, transcription and transposition of these elements should be greatly influenced by external factors such as biotic and abiotic stress conditions. To test this possibility, in vivo transcription of TLC1 was analyzed in stressed and nonstressed plants. In mature plants, Plant Physiol. Vol. 138, 2005

Figure 9. Histochemical analysis of GUS activity in leaves from transgenic plants harboring TG270::GUS or TRS1*/2*::GUS constructs. Histochemical analysis was carried out on leaves of transgenics plants exposed to mechanical wounding. After 18 h, leaves were collected and processed for histochemical analysis. C, Control leaves (without injuries); W, leaves exposed to wounding stress. 2081

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On the other hand, high salt and osmotic challenges have been reported to induce gene expression by both ABA-dependent and ABA-independent signal transduction pathways (Shinozaki and Yamaguchi-Shinozaki, 2000; Shinozaki et al., 2003). The latter appears to be the case for TLC1 expression, since ABA was unable to induce its transcription (Fig. 5). Recently, wounding and salt stresses have been described to activate the same response genes in Lycopersicon esculentum. Genes induced by wounding, such as Inh II, CDI, PS, and LOX coding for proteinase inhibitor II, cathepsin D inhibitor, prosystemin, and lipoxygenase, respectively, are also activated by high salt (Dombrowski, 2003). This regulation is not observed in the def-1 mutant, which is impaired in the octadecanoid pathway, thus suggesting the involvement of jasmonic acid in the response to saline stress (Dombrowski, 2003). Because TLC1 expression is activated by wounding and salt stresses and also by methyl jasmonate, it is likely that its regulation involves the jasmonic acid signaling cascade. The U3 Domain of TLC1.1 5# LTR Includes cis-Acting EREs

Nucleotide sequence analysis of 5# LTR revealed the features for the TLC1.1 promoter. Two 57-bp tandem repeats (TRS1 and TRS2) containing the sequence ATTTCAAA are present in the U3 domain (Fig. 6). This 8-bp element has been previously identified as an ethylene-responsive enhancer in the tomato E4 and carnation (Dianthus caryophyllus) GST1 genes, both responsive to ethylene. Footprinting analysis has indicated that this sequence corresponds to a specific transcription factor binding site (Montgomery et al., 1993; Itzhaki et al., 1994). The participation of these putative ERE boxes in the regulation of TLC1.1 expression was demonstrated through promoter mutation analyses. The low GUS expression observed in transient expression assays with the TRS1*, TRS2*, and TRS1*/2* mutants, together with the fact that these promoter derivatives were not able to respond to exogenous ethylene (Fig. 7), indicate that these sequences are functional regulatory elements and are responsible for the induction of TLC1.1 in response to ethylene. This assumption was confirmed by in vivo experiments with transgenic tobacco plants. Exogenous applied ethylene was sufficient to induce the expression of GUS in plants containing the wild-type promoter (P270::GUS), but not in plants containing the double ERE box mutant construct (PTRS1*/2*::GUS; Fig. 8). Furthermore, experiments with the same stable transgenic plants clearly showed that the ERE boxes are involved in the transcriptional activation by wounding stress (Fig. 9). These results suggest that an ethylene-dependent signaling is the principal transduction pathway involved in the regulation of the TLC1.1 retrotransposon. The induction of retrotransposon expression in response to stress conditions has been extensively ana2082

lyzed. The best studied are Tnt1 and Tto1 from tobacco, which are activated in response to several biotic and abiotic factors (Grandbastien et al., 1997; Takeda et al., 1999). While the expression of Tnt1 appears to be methyl jasmonate and salicylic acid dependent (Grandbastien et al., 1997; Mhiri et al., 1997; Beguiristain et al., 2001), the expression of Tto1 is only methyl jasmonate dependent (Takeda et al., 1999). It has been shown that expression levels of both tobacco retrotransposons are correlated with the number of tandem repeat sequences in the U3 domain (Grandbastien et al., 1997; Takeda et al., 1999). In the case of Tto1, it has been demonstrated that repeated 13-bp motifs are binding sites for a MYB-related transcription factor induced by wounding and elicitors (Sugimoto et al., 2000). The participation of ethylene in regulating several developmental processes as well as responses to biotic and abiotic stresses is well documented (for review, see Johnson and Ecker, 1998; Bleecker and Kende, 2000; Wang et al., 2002). Several studies have revealed the existence of ethylene-dependent signaling pathways activated in response to pathogen attack (Dong, 1998; Guo et al., 2000; Dıáz et al., 2002), wounding (O’Donnell et al., 1996; Watanabe et al., 2001), UV irradiation, and ozone exposure (Wang et al., 2002). In such cases, ethylene acts in a coordinated way with other signaling molecules such as salicylic acid and jasmonic acid, suggesting the existence of a complex network of regulatory pathways (Dong, 1998; Wang et al., 2002; O’Donnell et al., 2003; Guo and Ecker, 2004). Consequently, the residual GUS activity detected in protoplasts transformed with the single or double TRS mutant constructs (Fig. 8) and the histochemical signal observed in wounded TRS1*/2*::GUS plants (Fig. 9) could be explained by the existence of additional cis-elements in the TLC1.1 promoter that respond to other signal molecules. The elucidation of the molecular mechanisms, as well as the cross-talk regulation between the different signaling pathways mediating TLC1.1 expression, will be of central importance for understanding the activation of this retroelement under stress conditions and to determine the possible role of TLC1.1 in the adaptative response of L. chilense to the environmental challenges occurring in its natural habitat. MATERIALS AND METHODS Plant Material Lycopersicon chilense Dun. seeds were collected in the Atacama Desert of Chile. Seeds were surface sterilized with 10% (v/v) commercial bleach (0.5% [w/v] sodium hypochlorite NaOCl) and 0.05% (w/v) surfactant for 15 to 20 min, followed by three rinses with sterilized deionized water. Plants were cultured in vitro in Murashige and Skoog (MS) basal media (Murashige and Skoog, 1962) with 8 g L21 agar (pH 5.6–5.7) at 25°C in growth chambers under an 18/6-h photoperiod (120 mE m22). Four- to 6-week-old plants were used for experimentation. T1 transgenic tobacco (Nicotiana tabacum) plants were vegetatively propagated under sterile conditions, and many were transferred to soil and grown in a greenhouse. Others were cultured in vitro under the conditions described above in MS agar containing 100 mg mL21 kanamycin.



Primers The following oligonucleotides were used as primers for PCR and in vitro mutagenesis reactions: P1, 5#-GGAATTCTATGCTGACCAAGGTGGTAC-3#; P2, 5#-GGGGATCCTACCCTCCAAATGTGCTATAG-3#; P181, 5#-GGATCCCTGCTTCGAGCAGCGAC-3#; P182, 5#-GGAATTCTACTGGCAGGATCAAC-3#; L00, 5#-GGGAAGCTTGAGGAGTCCATCCACGAGAAC-3#; L270, 5#-GGGGATCCGATGCCTCACTCTTTTTTTCTCTCCTTTG-3#; L310, 5#-GGAAGCTTTGAGTTATATTGTAATGAGGTGG-3#; L578, 5#-GGGATCCGCTCTGATACCAGTTGTTGGG-3#; G300, 5#-GACCCACACTTTGCCGTAATG-3#; Mu1, 5#-TGAATAACTTTGTGCCACTGCAGAAGTTTGGTAGAGTTG-3#; Mu11, 5#-CAACTCTACCAAACTTCTGCAGTGGCACAAAGTTATTC-3#; Mu2, 5#-GAAAAATTTGTTTTTTGCCATCTAGAAGGTTTGGCAGAG-3#; Mu22, 5#-CTCTGCCAAACCTTCTAGATGGCAAAAAACAAATTTTT; Endo1, 5#-GAACGCGGGAATTGTTCG-3#; Endo2, 5#-GGAACATTCAACATACCACAA-3#; His-1, 5#-CCTAAATCTGCCAAGGCTGT-3#; His-2, 5#-GCCCTTTTAGCAGCAGGAGA-3#; Rbc1, 5#-CTCCTGAGTACCAAACCAAGGATACTG-3#; Rbc2, 5#-CATCCCAACAGGGGACGACCATAC-3#.

Probe Labeling Probes U5-PBS and PPT-U3 were obtained by PCR amplification of a pUC19 derivative containing the TLC1.1 retrotransposon using the primer mixtures L310/L578 and P1/P2, respectively. Probes for DNA genomic analysis of transformed plants were obtained by amplifying a 670-bp fragment corresponding to P270-GUS or PTRS1*/2*-GUS junctions from pBI270 and PBI1*/2*, respectively, using the primer mixture L00/G300. All probes were 32P-labeled by random priming using the Megaprime labeling kit (Amersham, Buckinghamshire, UK).

Genomic Library Screening A genomic library from L. chilense constructed in the lGEM12 vector (Promega, Madison, WI) was screened with a 300-bp probe corresponding to the reverse transcriptase domain of the TLC1 family of retrotransposons. The probe was obtained from the clone pTLC1.6 (Yan˜ez et al., 1998) and 32P-labeled by random priming using the Megaprime labeling kit (Amersham). Plaque hybridization was conducted as described by Sambrook et al. (1989). DNA from positive recombinant phages was purified by using the Wizard l DNA prep kit (Promega) according to the manufacturer’s instructions. The DNA insert was separated in a 0.7% agarose gel and transferred to Hybond-N nylon membranes (Amersham). Random priming 32P-labeled PPT-U3 and U5-PBS (Fig. 1) were used as molecular probes. Hybridization conditions were those described by Sambrook et al. (1989). The entire 6-kb EcoRI fragment was cloned into the respective site of pUC19.

Stress Treatment and RNA Extraction L. chilense plants were exposed to wounding, drought, and saline stress conditions. For wounding treatments, leaves were injured with Waugh tissue forceps. For salt stress, plants were cultured for 24 h in a one-fourth MS medium supplemented with 200 mM NaCl. Dehydration stress consisted in stopping watering the plants, and samples were collected after 30% of fresh weight was lost. Leaves from all treatments (300 mg) were processed for total RNA isolation with the SV RNA isolation kit (Promega), following the instructions provided by the manufacturer.

Chemical Treatments In vitro cultured L. chilense plants (4 weeks old) were sprayed with either 1 mM salicylic acid in sterile water/0.001% ethanol (pH 6.5 adjusted with KOH), 50 mM methyl jasmonate in sterile water/0.01% methanol, 100 mM ABA in sterile water/0.01% ethanol, 50 mM 2,4-D in sterile water/0.01% ethanol, or 1% v/v H2O2. Control plants were sprayed with water/0.01% ethanol/0.01% methanol. For ethylene treatments, plants were placed in sealed containers and then ethylene was taken from a concentrated stock (Alltech, Deerfield, IL) and injected into the containers using a syringe to give a final concentration of 10 mL L21. This concentration was monitored by gas chromatography (Clarus 500, Perkin Elmer, Cetus, Foster City, CA) every 3 h and remained stable throughout the treatment. Control plants were incubated in sealed containers without any chemical. The containers were opened after 6, 12, and 24 h, and


leaf samples (300 mg) were collected and processed for total RNA isolation as described previously. Transgenic tobacco plants were treated with ethylene for 18 h, and leaf samples were immediately collected and processed for fluorometric detection of GUS activity according to Jefferson et al. (1987).

Northern Hybridization Total RNA (10 mg) was separated in 1.5% agarose-formaldehyde gels and transferred to Hybond-N nylon membranes (Amersham). The U5-PBS and PPT-U3 DNA fragments (Fig. 1) were 32P-labeled and used as a probe. The probe for the large subunit of Rubisco was generated by PCR amplification from L. chilense genomic DNA using Rbc1 and Rbc2 as primers. The amplification product was sequenced and 32P-labeled by random priming. Hybridization and washing conditions were performed as described by Maimann et al. (2000).

RT-PCR Analysis RNA samples (5 mg) were reverse transcribed in a 20 mL reaction using the ThermoScript RT-PCR system for first-strand cDNA synthesis (Invitrogen, Carlsbad, CA). Two-microliter aliquots from this reaction were amplified by PCR using the corresponding oligonucleotides as primers. PCR amplification with Taq DNA polymerase was performed in a 30-cycle reaction under the following conditions: 94°C (45 s), 55°C (30 s), 72°C (60 s).

Construction of LTR-GUS Fusions Specific domains or the complete 5# LTR of TLC1.1 were obtained by PCR amplification using pTLC2500 as a template, which corresponds to a pUC19 derivative clone containing the first 1,924 bp (5# moiety) of the TLC1.1 retrotransposon. The primer mixtures L00/L578, L00/L270, and L310/L578 were used to amplify the complete LTR, the U3-R domain, and the U5 region, respectively, introducing a target site for BamHI and HindIII at the end of each amplified fragment. PCR reactions were conducted as described previously, except that high-fidelity Pfu DNA polymerase was used for amplification. The pBI221 vector (CLONTECH Laboratories, Palo Alto, CA) containing the 35SCaMV-GUS construct was digested with HindIII and BamHI to remove the 35S CaMV promoter, and the amplification products double digested with the same endonucleases were ligated into the promoterless vector. The chimeric constructs were named pTG578 and pTG270, respectively.

In Vitro Mutagenesis The chimeric plasmid pTG270 was used as template for in vitro mutagenesis of the ERE box motifs present in the TRS1 and TRS2 repeats. The QuikChangeSite-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used according with the instructions provided by the manufacturer. Primers Mu1 and Mu11 were used for mutagenesis of the ERE box at TRS1 generating the mutant pTRS1*, which includes a PstI recognition site. The ERE box in TRS2 was mutagenized using primers Mu2 and Mu22, yielding the mutant pTRS2*, which contains an XbaI target sequence. The double mutant, pTRS1*/2*, was obtained in a second round of mutagenesis with primers Mu2 and Mu22 over the pTRS1* mutant. The screening of mutants was performed by PstI, XbaI, or PstI/XbaI digestion and confirmed by DNA sequencing.

Transient Expression Assays L. chilense leaves from in vitro cultured plants (4–6 weeks old) were processed for protoplasting as described by Chupeau et al. (1974). Briefly, leaf tissue was treated with 1,100 mg/L Onozuka-R10 cellulase (Yakult Biochemicals) and 250 mg/L Macerozyme-R10 (Yakult Biochemicals, Tokyo) during 16 h at 22°C in 0.4 mM mannitol, 4 mM CaCl2, 2.6 mM MES (pH 5,6). After enzymatic digestion, viable protoplasts were purified by washing in 0.2 M CaCl2 (three times) and two sequential steps of floating in Suc cushions of 21% and 16% Suc, respectively. For electroporation, 1 3 106 protoplasts were placed in a 0.4-cm chamber and mixed with a solution containing 10 mg of plasmid DNA, 50 mg of sonicated salmon sperm carrier DNA, 0.4 mM mannitol, 4 mM CaCl2, 90 mM KCl, 10 mM HEPES (pH7,0). DNA transfer was achieved by three electric pulses (300 V, 150 mF, 20V) in a Gene Pulser II electroporator (Bio-Rad). After electroporation, protoplasts were incubated in MS medium for 48 h at 22°C in darkness. For ethylene treatment, protoplast in

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MS medium were placed into a sealed container and ethylene was taken from a concentrated stock (Alltech) and injected trough a valve into the containers using a syringe to give a final concentration of 10 mL L21. Treated protoplasts were incubated 48 h at 22°C in the dark with continuous shaking. The ethylene concentration was monitored by gas chromatography (Clarus 500, Perkin Elmer, Cetus) every 3 h and remained stable throughout the treatment. Control protoplasts were incubated in the same conditions but an equal volume of air was injected. Cultured protoplasts were lysed in 10 mM EDTA, 10 mM dithiothreitol, 50 mM sodium phosphate (pH 7.0) by successive freezing and thawing steps and strong shaking. The protein concentration in the extract was estimated according to Bradford (1976) and GUS activity was measured as described by Jefferson et al. (1987).

Plant Transformation The plasmids pTG270 and pTRS1*/2* were used to obtain the fragments TG270 and TRS1*/2* by digestion with HindIII and BamHI. These fragments were ligated into the promoterless binary vector pBI121 (CLONTECH Laboratories), which had been previously digested with the same restriction enzymes to remove 35S CaMV and purified. Thus, GUS gene reporter remained under the control of the P270 or PTRS1*/2* promoters. The plasmids generated were named pBI270 and pBI1*/2*. These plasmids were transferred to Agrobacterium tumefaciens strain LBA4404 (Gibco BRL, Carlsbad, CA) by electroporation, following the instructions provided by the manufacturer. Afterward, leaf discs of sterile-grown tobacco (var Xanthi) were transformed, essentially as described by Horsch et al. (1984). Transformed plants were selected by rooting several times on kanamycin-containing medium and transferred to compost. Presence and integrity of T-DNA insertions were determined by PCR using GUS- and promoter-specific primers. Insertion copy number was determined by Southern-blot analysis of genomic DNA from transformed plants digested with either HindIII or EcoRI endonucleases to generate T-DNA/plant DNA junction fragments. Digested DNA was separated in a 0.8% agarose gel and transferred to Hybond-N nylon membranes (Amersham) and hybridized with a random priming 32P- labeled 620-bp fragment containing either the P270-GUS or the PTRS1*/2*-GUS junctions as molecular probes. Hybridization conditions were as described by Sambrook et al. (1989). For segregation analysis of kanamycin resistance and growth of T1 plants, primary transformants (T0) were vegetatively propagated under sterile conditions and transferred to soil and grown in a greenhouse. Greenhouse plants were allowed to self-fertilize and seeds were collected.

GUS Histochemistry Histochemical analyses of GUS activity were performed on T1 transgenic plants harboring a single copy of the insert. Leaves from 4-week-old in vitro grown plants were subjected to mechanical wounding as described above. Leaves were collected 48 h after injury for histochemical analysis. Histochemistry was performed by placing whole leaves in the appropriate staining buffer containing X-Gluc (0.5 mg mL21 5-bromo-4-chloro-3-indolyl-b-Dglucuronide [Duchefa, Haarlem, The Netherlands] in 50 mM sodium phosphate, pH 7, 0.05% Triton X-100, 0.1 mM K4Fe(CN)6.3H2O, 0.1 mM K3Fe(CN)6) and incubated in the dark at 37°C for 16 h (Jefferson et al., 1987). Staining solution was infiltrated under reduced pressure for 2 to 5 min. After staining, leaves were cleared of chlorophyll by washing with methanol/acetone solutions (3:1, v/v), preserved in glycerol (100%), and photographed with a stereo microscope.

Statistical Analysis GUS activity in the presence or absence of ethylene in either unwounded or wounded plants (Fig. 8, A and B) was compared by performing a one-way ANOVA followed by multiple comparisons with Tukey’s honestly significant difference (HSD) mean-separation test. Additionally, a three-way ANOVA followed by multiple comparisons with Tukey’s HSD mean-separation test was also performed analyzing together the effect of genotype, ethylene presence, and wounding on GUS activity. All statistical analyses were performed using STATISTICA 6.0 (www.statsoft.com). Since data showed normal distribution and homocedasticity, no data transformation was performed.

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Sequence data from this article have been deposited with the EMBL/ GenBank data libraries under accession number AF279585.

ACKNOWLEDGMENTS We thank Dr. Albert Boronat (Departamento de Bioquı´mica y Biologıá Molecular, Universidad de Barcelona) and Dr. Salome´ Prat (Departamento de Gene´tica Molecular de Plantas, Centro Nacional de Biotecnologıá, Consejo Superior de Investigaciones Cientı´ficas, Madrid) for critical and stimulating discussions. We also thank Dr. Josep Casacuberta (Departamento de Gene´tica Molecular, Centro de Investigacioń y Desarrollo, Consejo Superior de Investigaciones Cientı´ficas, Barcelona), Dr. Hugo Penã-Corte´s (Centro de Biotecnologıá, Universidad Federico Santa Marıá, Valparaı´so-Chile), and Dr. Jose´ Casaretto (Instituto de Biologıá Vegetal y Biotecnologıá, Universidad de Talca) for critical reading of the manuscript, and Dr. Claudio Ramirez (Instituto de Biologıá Vegetal y Biotecnologıá, Universidad de Talca) for help with the statistical analysis. Received January 17, 2005; revised May 10, 2005; accepted May 10, 2005; published July 22, 2005.

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