Involvement of ethylene in stress-induced expression of the TLC1.1 retrotransposon from Lycopersicon chilense Dun

Abstract

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 stress-associated 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 beta-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 ethylene-induced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the beta-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.

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.

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 M_r marker (Invitrogen).

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.

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 endo1- and 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 M_r standard (Promega).

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 μg 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 α-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 μm methyl jasmonate, 100 μm ABA, 50 μm 2,4-D, or 1% v/v H₂O₂. Control plants were sprayed with water/0.01% ethanol/0.01% methanol.

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.

Figure 7.

Transient expression analysis in L. chilense leaf protoplasts. Protoplasts were trasnsformed with 10 μg 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.

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 μL L⁻¹ 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 ± se.

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.