Lettuce Polyubiquitin Promoter-terminator Promotes Transgene Expression Transcriptionally in Lettuce and Translationally in both Lettuce and Arabidopsis

Abstract

The Cauliflower mosaic virus 35S promoter (P35S) induces transgene expression with insufficient activity and stability in some plant species, including lettuce. To develop a system to provide sufficient gene expression, a polyubiquitin promoter (PLsUbi) and terminator (TLsUbi) were isolated from lettuce, and this system was functionally compared with the conventional P35S-NOS terminator (P35S-Tnos) system by using a β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis, PLsUbi induced higher GUS activity than P35S, and the PLsUbi-TLsUbi combination induced higher GUS activity compared with the PLsUbi-Tnos combination, suggesting that the polyubiquitin terminator promotes transgene expression in concert with PLsUbi. The PLsUbi-TLsUbi combination induced less accumulation of GUS mRNA but > 10-fold higher GUS enzyme activity than the P35S-Tnos combination, suggesting that the PLsUbi-TLsUbi combination translationally promoted GUS expression in Arabidopsis. In transgenic lettuce, PLsUbi-TLsUbi transcriptionally and translationally promoted GUS expression, inducing approximately 16-fold-higher accumulation of GUS mRNA and > 800-fold-higher GUS enzyme activity compared with those induced by P35S-Tnos. Bisulfite sequencing methylation analysis of the introduced promoter sequences indicated that, for PLsUbi, the mean percentage of methylated cytosines in lettuce was 3.5 times that in Arabidopsis. For P35S, the mean percentage of methylated cytosines in lettuce was > 10 times that in Arabidopsis, and this methylation may be a major reason underlying the transcriptional inactivation of P35S in lettuce. Together, our results indicate that PLsUbi-TLsUbi promotes transgene expression in lettuce and Arabidopsis and may have broad applications in genetic engineering of additional plant species.

Introduction

Lettuce (Lactuca sativa L.) is one of the most important vegetable crops worldwide. Although extensive lettuce crossbreeding has improved many traits (including yield, tolerance to biotic and abiotic stress, quality, and shelf life), transgenic technology is especially useful in improving characteristics that are difficult to introduce by crossbreeding from genetic resources or breeding materials. Many attempts have been made to introduce practically important traits into lettuce through transgenic approaches; these traits include virus resistance (Kawazu et al., 2016), salt and drought tolerance (Kim et al., 2013), herbicide resistance (Nagata et al., 2000), and increased contents of certain compounds (Lee et al., 2007; Sun et al., 2006). Biodiversity risk assessments of transgenic lettuce have also been reported, with the aim of achieving practical use of transgenic plants (Giannino et al., 2008; Goto et al., 2001; Kawazu et al., 2010).

To improve crops through transgenic technology, appropriate use of gene expression systems comprising a promoter and a terminator is crucial. The system combining the Cauliflower mosaic virus 35S promoter (P35S) and Agrobacterium nopaline synthase gene terminator (Tnos) has frequently been used to induce constitutive and strong gene expression in transgenic plants. However, the activity and stability of this promoter-terminator system is inadequate in certain situations, depending on the host plant species, developmental stage, tissue type, and generation after gene introduction events (Mishiba et al., 2005; Sun et al., 2006; Sunilkumar et al., 2002). The expression of P35S-driven transgenes in lettuce has often been reported to be inadequate and frequently inactivated in successive generations. For example, McCabe et al. (1999) produced transgenic lettuce carrying a bar gene for herbicide resistance, driven by either P35S or the 784-bp plastocyanin promoter from a pea (petE). Among kanamycin-resistant P35S-bar- and petE-bar-transformed T₀ plants, herbicide resistance was found in only 11% of P35S-bar T₀ plants compared with 75% of petE-bar T₀ plants. Moreover, only 2.5% of kanamycin-resistant P35S-bar T₀ plants transmitted herbicide resistance at a high frequency to the T₃ seed generation, as compared with 97% of kanamycin-resistant petE-bar plants (McCabe et al., 1999). Sun et al. (2006) produced transgenic lettuce with a P35S-driven gene encoding a taste-modifying protein, miraculin. However, the expression of P35S-driven miraculin was much lower in T₁ or T₂ lettuce plants than in T₀ plants. Thus, P35S has often been shown to be inadequate in terms of gene expression in lettuce.

To develop a system to provide high, stable transgene expression in lettuce, we considered a polyubiquitin gene promoter. Ubiquitin is a highly conserved eukaryotic protein involved in protein degradation. Polyubiquitin genes encode polyproteins comprising several tandem repeats of the ubiquitin-coding unit (Sullivan et al., 2003). Polyubiquitin gene promoters have been isolated from many higher plant species including rice (Bhattacharyya et al., 2012), maize (Christensen et al., 1992), soybean (Hernandez-Garcia et al., 2009), potato (Garbarino et al., 1995), sugarcane (Wei et al., 2003), and Arabidopsis thaliana (Callis et al., 1990). These promoters have been shown to provide strong constitutive expression; therefore, they may hold promise in lettuce transgenic expression systems.

In the present study, we report the isolation and functional analysis of a promoter (PLsUbi) and terminator (TLsUbi) from a lettuce polyubiquitin gene. During the study, we provided the PLsUbi and TLsUbi cassette to Hirai et al. (2011), who used it to express the miraculin gene in lettuce. They compared the expression levels of the P35S-driven and polyubiquitin-promoter-driven miraculin gene in transgenic lettuce and found the expression of the polyubiquitin-promoter-driven miraculin to be higher and more genetically stable. In our study, a β-glucuronidase (GUS) reporter gene was used for functional analysis of PLsUbi and TLsUbi. Gene expression analysis was performed in both transgenic lettuce and transgenic Arabidopsis. In addition, four promoter-terminator combinations based on PLsUbi, TLsUbi, P35S, and Tnos were analyzed in transgenic Arabidopsis. DNA-methylation analyses of PLsUbi and P35S were also performed to determine the relationship between promoter methylation rates and transgene expression. PLsUbi-TLsUbi induced higher reporter-gene expression than P35S-Tnos by enhancing transcription in lettuce and enhancing translation in both lettuce and Arabidopsis. By improving transgene expression, the PLsUbi-TLsUbi system may facilitate lettuce crop improvement and may be further applicable to other plant species.

Materials and Methods

Cloning of lettuce polyubiquitin promoter and terminator

Complementary DNA (cDNA) sequences encoding lettuce polyubiquitin were retrieved from the DFCI Gene Index Databases (ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/), LsGI release 2. Based on the number of assembled expressed sequence tags (ESTs), the unigene sequence TC8119 was selected. Cloning of the 5'-upstream region flanking the TC8119 sequence was performed with a thermal asymmetric interlaced PCR (TAIL-PCR) method (Liu and Whittier, 1995) using the TC8119-specific primers LsUbi-5GSP1 (5'-TCCGG CCGTC CTCGA GTTGC-3'), LsUbi-5GSP2 (5'-TGTCT TGGAT TTTCG CCTTG-3'), and LsUbi-5GSP3 (5'-TTTTC GCCTT GACGT TGTCT-3') for the first, second, and third TAIL-cycling, respectively (Fig. 1). The 3' downstream region flanking the TC8119 sequence was isolated through a DNA walking method described by Siebert et al. (1995), with a GenomeWalker Universal Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and the TC8119-specific primers LsUbi-3GSP1 (5'-GCCGG AAAGC AGCTG GAGGA TG-3') and LsUbi-3GSP2 (5'-TTGGT GGTGT TATGA AGGTT GTTGA-3') for the first and second PCR, respectively. Amplified DNA fragments were cloned into the pGEM-T vector (Promega, Madison, WI, USA) and sequenced with BigDye v3 sequencing premix and a 3730xl DNA sequencer (Applied Biosystems, Foster City, CA, USA). Based on the sequences of the primary clones, the primer pair LsUbiPro-Sph-U (5'-CTCGC ATGCG AAACA AGTGT CCGAA ATCCT-3') and LsUbiPro-Sma-L (5'-CTCCC CGGGC TGTTA AAAAA AGAAC GAAAC-3') was designed for amplification of an appropriate length of the DNA fragment for the promoter, and 5'-SphI and 3'-SmaI sites were added to allow for insertion of the fragment in front of the GUS reporter gene. In the same manner, the ubiquitin terminator fragment with 5'-SacI and 3'-EcoRI sites was obtained with the primers Tubi-Sac-U (5'-CTCGA GCTCT GGTTT GGTGG TGTTA TG-3') and Tubi-Eco-L (5'-CTCGA ATTCA ACGCG GGCTA TAATC TA-3').

Fig. 1

Structure of the cloned polyubiquitin promoter PLsUbi and terminator TLsUbi from lettuce. The thick lines indicate the sequence of TC8119, which lacks the intron because the sequence was derived from ESTs. 5GSP and 3GSP indicate the binding sites of primers used for cloning the 5'-upstream and 3'-downstream regions flanking the TC8119 sequence, respectively.

Vector construction and plant transformation

The binary vector pZK3B was kindly provided by Dr. M. Kuroda of the Central Region Agricultural Research Center, NARO, Japan (Kuroda et al., 2010). The vector was a derivative of pPZP202 (Hajdukiewicz et al., 1994) containing the neomycin phosphotransferase II gene driven by the NOS promoter as a selectable marker and a multiple-cloning site for integration of the gene cassette of interest. The transgenes consisting of a GUS gene under control of four promoter–terminator combinations were constructed (Fig. 3a), inserted into pZK3B and introduced into Agrobacterium tumefaciens strain C58C1. Lettuce (cv. Watson) transformation was done according to Kawazu et al. (2006). ‘Floral dip’ transformation of Arabidopsis (ecotype Columbia) was performed according to Clough and Bent (1998). Independent T₀ plants were grown in a containment greenhouse and self-pollinated, and T₁ seeds were harvested. To standardize the developmental stages, all experiments except for histochemical GUS staining were performed on two-week-old seedlings in the T₁ generation. The T₁ seeds were aseptically sown on Murashige and Skoog (MS) agar plates containing 50 mg·L⁻¹ kanamycin, and plants growing normally on the selectable medium were collected. For each transgenic line, samples from at least 20 individuals were pooled before protein and nucleic acid extraction to minimize the influence of transgene segregation among T₁ plants. Harvested plant samples were frozen in liquid nitrogen, pounded into powder with a mortar and pestle, and divided into aliquots for each experimental process for protein, DNA, and RNA extraction.

GUS assay

Histochemical GUS staining and fluorometric quantitative GUS assays were done according to Kosugi et al. (1990). Crude protein concentrations were determined with a Quick Start Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using bovine serum albumin as a standard. Spectrophotometric and fluorophotometric measurements were performed in at least triplicate with an ARVO (VICTOR) plate reader (PerkinElmer, Inc., Waltham, MA, USA).

Quantitative real-time PCR (qPCR)

Total RNA samples were isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), treated with DNase with a TURBO DNA-free Kit (Applied Biosystems/Ambion, Austin, TX, USA), and then purified with an RNeasy Mini kit (Qiagen, Hilden, Germany). The quantity and quality of the isolated total RNA samples were examined with an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), and 5 μg of total RNA was used for reverse transcription with random hexamers by using a SuperScript First-Strand Synthesis System (Invitrogen). The qPCR for GUS mRNA was performed in triplicate in a total volume of 10 μL containing 2 μL of 10×-diluted cDNA, 2 μM of each primer (qGUS-F: 5'-CACAG CCAAA AGCCA GACAG AG-3' and qGUS-R: 5'-CGACC AAAGC CAGTA AAGTA GAACG-3') and 5 μL of SYBR Premix Ex Taq (Takara Bio, Inc., Shiga, Japan) on a LightCycler480 real-time PCR instrument (Roche Applied Science, Mannheim, Germany). The lettuce (DDBJ/EMBL/GenBank accession number AY260165) and Arabidopsis (DDBJ/EMBL/GenBank accession number AK230311) actin genes were used as the endogenous references for each species. The primer pairs for the reference genes were LsACT-F (5'-AGGGC AGTGT TTCCT AGTAT TGTTG-3') and LsACT-R (5'-CTCTT TTGGA TTGTG CCTCA TCT-3') for lettuce, and AtACT2-F (5'-ACAGT GTCTG GATCG GTGGT TC-3') and AtACT2-R (5'-CCCCA GCTTT TTAAG CCTTT GATC-3') for Arabidopsis. The relative GUS mRNA amount was calculated as the ratio of the quantity of PCR product of the GUS gene over that of the reference gene.

DNA-methylation analysis

For analysis of DNA methylation by bisulfite sequencing, genomic-DNA preparation and bisulfite treatment were carried out with a DNeasy Plant Mini Kit (Qiagen) and EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s instructions. PCR amplification of the targets and cloning of the amplified products were performed according to Kanazawa et al. (2007) with the following PCR primer pairs: P35Sbs–346F1 (5'-TATTG AGATT TTTTA ATAAA GGGTA A-3') and P35Sbs+1R1 (5'-TCCTC TCCAA ATAAA ATAAA CTTC-3') for P35S first-round PCR, and P35Sbs–323F2 (5-TAATA TTTGG AAATT TTTTT GGATT-3') and P35Sbs-21R2 (5'-TTCCT TATAT AAAAA AAAAA TCTTA C-3') for P35S second-round PCR. For selective amplification of the introduced ubiquitin-promoter sequence, the following forward primers and reverse primers were designed based on the promoter and the GUS gene: PUBi-BSA-F1 (5'-TATTT TTAAT ATTAA TAATA TATTT-3') and GUS-BSA-R1 (5'-CTACA AAACA TAACA TAAAA AACTA-3') for PLsUbi-GUS first-round PCR, and PUBi-BSA-F2 (5'-ATTTA AAATT AAATA TTTTA TTGAT-3') and GUS-BSA-R2 (5'-AAACA TAACA TAAAA AACTA ACCAC-3') for PLsUbi-GUS second-round PCR. The PCR products were cloned into the pGEM-T Easy Vector (Promega), and for each transgenic plant line, 32 clones were randomly picked and sequenced.

Results

Cloning of the polyubiquitin gene promoter and terminator in lettuce

From the DFCI Gene Index Databases, six lettuce unigenes were identified that showed high sequence similarity to known plant polyubiquitin genes. Each unigene sequence resulted from clustering and assembly of ESTs, and the largest number of 129 ESTs was assembled into unigene sequence TC8119. The second-largest assembly was TC12394, which was assembled from 17 ESTs. The deduced amino acid sequence from the putative open reading frame (ORF) encoded in TC8119 corresponded to a complete pentameric polyubiquitin and was 100% identical to sequences of other plant ubiquitin monomers such as those encoded by Arabidopsis AtUbi3 (Callis et al., 1995). Postulating that the number of ESTs assembled into the respective unigenes reflected the unigene expression level in vivo, we isolated flanking genomic-DNA regions of TC8119 as a candidate promoter and terminator to induce constitutive and strong gene expression. A TAIL-PCR method (Liu and Whittier, 1995) was used to clone the 5'-upstream region flanking the TC8119 sequence: the resulting 3-kb genomic-DNA fragment we isolated contained a nucleotide sequence identical to the 5'-end region of TC8119. The region corresponding to the TC8119 sequence included a putative 5' untranslated region (UTR) 457-bp intron (Fig. 1). By comparing the TC8119 and the cloned genomic sequences, we confirmed that the invariant sequences found in all plant polyubiquitin genes (Samadder et al., 2008), CAAG/gta at the 5' splice site and cag/ATG at the 3' splice site of the 5'-UTR intron, were also conserved in this clone. The start codon (ATG) of the ORF was found to be adjacent to the junction of the first intron and the second exon, an arrangement commonly present in the known plant polyubiquitin genes (Binet et al., 1991; Sivamani and Qu, 2006). Assuming that the nucleotide position corresponding to the 5' end of the TC8119 sequence was the transcription-initiation site (+1), we found a TATA box at position (−33), an arrangement similar to those of the other plant polyubiquitin promoters.

To clone the 3'-downstream region flanking the TC8119 sequence, we used a DNA walking method (Siebert et al., 1995), and a 1-kb genomic DNA fragment was isolated from the DraI-digested GenomeWalker library. The fragment contained a 262-bp sequence identical to the 3'-end sequence of the TC8119 ORF. A putative polyadenylation signal AAUUAAA (Wickens and Stephenson, 1984) was found in the adjacent sequence immediately downstream of the 3' end of the TC8119 sequence.

Based on the sequences of the isolated fragments, 1908-bp and 598-bp genomic-DNA fragments which corresponded to the upstream and downstream flanking regions of the TC8119 ORF, respectively, were amplified de novo by genomic PCR and inserted into the cloning vector. The nucleotide sequences of the clones were confirmed by comparison with the results of direct sequencing of the corresponding PCR products to avoid selecting clones with undesired nucleotide mutations. Finally, 1908 bp and 598 bp were denoted PLsUbi and TLsUbi, respectively, and used for further analysis. The nucleotide sequences have been deposited in the DDBJ/EMBL/GenBank database under accession numbers AB500086 (PLsUbi) and AB500087 (TLsUbi).

Analysis of promoter and terminator function based on GUS reporter activity

In preliminary experiments, the PLsUbi::GUS::Tnos construct or P35S::GUS::Tnos construct was introduced into Arabidopsis and lettuce, and GUS activity was detected through histochemical GUS assays (Fig. 2). Strong GUS activity (blue staining) was detected in transgenic lettuce seedlings with PLsUbi::GUS::Tnos (Fig. 2a), but not those with P35S::GUS::Tnos (Fig. 2b). Strong GUS activity was detected in transgenic Arabidopsis with PLsUbi::GUS::Tnos (Fig. 2c and d), and the activity was stronger in leaves with PLsUbi::GUS::Tnos (Fig. 2d) compared to P35S::GUS::Tnos (Fig. 2e). These results suggest that the PLsUbi obtained in this study possesses strong and constitutive promoter activity.

Fig. 2

GUS expression in transgenic lettuce seedlings of T₁ generation (a, b) and in inflorescence (c) and mature leaves (d, e) of transgenic Arabidopsis of T₀ generation. The fusion genes PLsUbi::GUS::Tnos (a, c, d) or P35S::GUS::Tnos (b, e) were introduced via Agrobacterium-mediated transformation.

For further analysis, GUS expression in Arabidopsis was compared among four promoter-terminator combinations: P35S-Tnos, P35S-TLsUbi, PLsUbi-Tnos, and PLsUbi-TLsUbi (Fig. 3a). After these constructs were introduced into Arabidopsis, GUS expression in transgenic T₁ seedlings was determined through a fluorometric, quantitative GUS assay (Fig. 3b). In transgenic Arabidopsis, the mean GUS activity induced by PLsUbi-Tnos was 7.4 times that induced by P35S-Tnos (22.1 and 3.0, respectively; Fig. 2b); therefore, PLsUbi promoted gene expression to a greater extent than P35S. GUS activity induced by P35S-TLsUbi was approximately the same as that induced by P35S-Tnos, while that induced by PLsUbi-TLsUbi was the highest at about 3.1 times that of PLsUbi-Tnos (69.3 and 22.1, respectively). This result shows that TLsUbi, when acting in concert with PLsUbi, promotes transgene expression.

Fig. 3

Transgene construction and reporter-gene expression in transgenic plants. (a) The four promoter-terminator combinations introduced into Arabidopsis. (b) GUS enzyme activity measured in T₁ Arabidopsis seedlings. Four, six, six and seven independent T₁ plants were examined for P35S-Tnos, P35S-TLsUbi, PLsUbi-Tnos, and PLsUbi-TLsUbi constructs, respectively. Each open circle indicates the GUS activity of a plant (pmol 4MU·μg⁻¹ protein·min⁻¹), and horizontal bars indicate the averages. Different superscript letters (a–c) indicate significant differences (P < 0.05) on the basis of Steel-Dwass multiple comparison. The vertical axis is a log scale.

Relationship between GUS activity and GUS mRNA accumulation in transgenic plants

To estimate the contribution of translational regulation to the expression of a transgene under control of the PLsUbi-TLsUbi system, we examined the relationship between GUS enzyme activity and GUS mRNA accumulation in transgenic Arabidopsis and lettuce plants with PLsUbi-TLsUbi or P35S-Tnos (Table 1). For each of the four combinations of constructs and species, four or five independent T₁ transgenic plants were used for the analysis. GUS mRNA accumulation was standardized to the amount of actin mRNA. In Arabidopsis, mean GUS enzyme activity for PLsUbi-TLsUbi was 12.7 times that of P35S-Tnos (60.9 and 4.8, respectively), although the mean GUS mRNA accumulation for PLsUbi-TLsUbi was only 43% that of P35S-Tnos (29.6 and 68.9, respectively). The mean translation-index values (i.e., the ratio of GUS activity to mRNA accumulation) for PLsUbi-TLsUbi were more than 30 times those of P35S-Tnos (2.3 and 0.07, respectively). These differences were significant at the 5% level.

Table 1

GUS activity, relative GUS mRNA accumulation and methylation of promoter in transgenic plants.

In lettuce, mean GUS mRNA accumulation for PLsUbi-TLsUbi was 16 times that of P35S-Tnos (3.2 and 0.2, respectively). The mean translation-index values for PLsUbi-TLsUbi were approximately 39 times those of P35S-Tnos (10.9 and 0.28, respectively). These differences were significant at the 5% level.

Methylation analysis of the introduced promoter sequences in transgenic plants

Methylation status in the introduced promoter was examined through bisulfite sequencing, and the percentage of methylated cytosines was calculated for both P35S and PLsUbi in transgenic lettuce and Arabidopsis (Table 1). For PLsUbi, the mean percentage of methylated cytosines in lettuce was 3.5 times that in Arabidopsis (16.6% and 4.8%, respectively) although the difference was not significant at the 5% level. To compare PLsUbi-driven GUS mRNA accumulation in lettuce and Arabidopsis, we standardized mRNA accumulation to the amount of total RNA (Table 1, c): mean accumulation in lettuce was only 35% that in Arabidopsis (2.4 and 6.9, respectively), and the difference was significant at the 5% level. In lettuce, the percentages of methylated cytosines in Line U-L2 (33.8%) and U-L3 (34.1%) were higher than those in Line U-L1 (2.4%) and U-L5 (2.3%), while GUS mRNA accumulation in Line U-L2 (1.5) and U-L3 (0.3) was lower than that in Line U-L1 (5.6) and U-L5 (3.0). These results suggest that higher methylation rates were associated with lower PLsUbi activity.

For P35S, the mean percentage of methylated cytosines in lettuce was 11.7 times that in Arabidopsis (30.4 and 2.6, respectively). Only 1.8–3.3% of cytosine residues were methylated in Arabidopsis, whereas 19.6–37% of cytosine residues were methylated in lettuce. Furthermore, cytosine residues in the two TGACG motifs of the activation sequence 1 (as-1) site, which is important for P35S activity (Lam et al., 1989), had approximately 29-fold-higher methylation rates in transgenic lettuce (66.4) than in Arabidopsis (2.3); Table 1 and Fig. 4. GUS mRNA accumulation, as standardized to the amount of total RNA, was much lower in lettuce, at 0.7%, that in Arabidopsis (0.1 and 14.4, respectively). These differences were significant at the 5% level. The results strongly suggest that P35S is preferentially DNA-methylated in lettuce. This higher methylation rate may be a major reason for the transcriptional inactivation of P35S in lettuce.

Fig. 4

(a, b) Average percentages of methylcytosine of all cytosines in the P35S sequence in the transgenic Arabidopsis (a) and lettuce (b) lines. The vertical bars show the average frequencies of methylcytosine per sequenced clone in each transgenic line. The analyzed sequences cover the −298 to −48 region of the promoter and are indicated as the horizontal axes. The positions of cytosine residues in the two TGACG motifs of the as-1 site are indicated by arrowheads.

Discussion

In the present study, we demonstrated that PLsUbi-TLsUbi, compared with P35S-Tnos, induced higher expression of a GUS reporter gene, through transcriptional promotion in lettuce and translational promotion in both lettuce and Arabidopsis. PLsUbi induced higher transgene expression when it was combined with TLsUbi, but the underlying mechanism remains to be determined.

The accumulation of GUS mRNA was different among transgenic lines even though combinations of constructs and species were the same (Table 1). For example, relative GUS mRNA accumulation in Line U-L1 was 8.3, while the accumulation in Line U-L3 was 0.3. The difference could be related to the copy number of the transgene. Hirai et al. (2011) checked the copy number of the P35S-driven or PLsUbi-driven miraculin gene in transgenic lettuce. Out of 16 transgenic lettuce plants with the P35S-driven miraculin gene, nine carried one copy, four carried two copies and three carried multicopies of the miraculin gene. Miraculin gene expression was observed in 10 plants, but three plants with two copies and three plants with multicopies showed no expression. Out of 22 transgenic lettuce plants with the PLsUbi-driven miraculin gene, six carried one copy, nine carried two copies and seven carried multicopies of the miraculin gene. Miraculin gene expression was observed in 20 plants, but two plants with multicopies showed no expression. In our study, the copy number of the transgene was unknown, but it is possible that transgenic plants with lower levels of GUS gene expression had higher copies of the transgene.

The part of PLsUbi-TLsUbi responsible for the translational enhancement remains unknown, but the 5'-UTR intron of PLsUbi is a candidate. In support of this possibility, Kamo et al. (2012) reported that the 5'-UTR intron of the Gladiolus polyubiquitin promoter enhances the translation efficiency of a GUS reporter gene in Gladiolus and Arabidopsis. Samadder et al. (2008) observed transcriptional and post-transcriptional enhancement in rice by the rice polyubiquitin rubi3 gene, and they attribute this enhancement to the 5'-UTR intron that is commonly found in plant polyubiquitin genes, as well as PLsUbi at a conserved location immediately 5' of the initiator Met codon. Moreover, Norris et al. (1993) found that in Arabidopsis, gene expression with the Arabidopsis polyubiquitin UBQ10 promoter without the 5' intron was lower than that with the 5' intron. Intron-mediated enhancement of gene expression has been reported at both the transcriptional and translational levels (Bourdon et al., 2001; Rose, 2004), and has also been widely observed in eukaryotes including plants, mammals, insects, fungi, and nematodes (Rose, 2008), although the underlying mechanism remains unknown. Therefore, the 5'-UTR intron in PLsUbi may have translationally enhanced GUS expression in our study.

Methylation, another factor known to affect transgene expression, may explain the inadequate expression conferred by P35S. Okumura et al. (2016) produced transgenic lettuce with a P35S-driven or modified P35S-driven sGFP gene and reported that the extent of the methylation in P35S correlated well with sGFP mRNA suppression in lettuce. Our results also indicated a higher percentage of methylated cytosines in P35S and a lower level of P35S-driven GUS mRNA accumulation in lettuce than in Arabidopsis. These results together suggest that, in lettuce, P35S is preferentially subject to DNA methylation, and this methylation may underlie the transcriptional inactivation of the P35S-driven transgene in lettuce.

One possible mechanism for this methylation-driven inactivation may involve cellular factor activation sequence factor 1 (ASF-1), originally identified in peas and tobacco, which binds as-1, a region of P35S containing two TGACG motifs. Lam et al. (1989) reported that mutations at these motifs inhibit binding of ASF-1 to P35S and decrease expression of a P35S-driven transgene. Kanazawa et al. (2007) have since found that methylation of cytosine residues in CpG dinucleotides within the −91 to +1 fragment of P35S, including as-1, strongly inhibits binding of a nuclear factor in vitro. In our study, 66% of cytosines at as-1 were methylated, on average, suggesting that methylation inhibited binding of ASF-1 to P35S and decreased the accumulation of P35S-driven GUS mRNA in lettuce.

Given the inhibitory effects of methylation on P35S-driven expression, we investigated the effects of methylation on expression driven by our polyubiquitin-based system by comparing PLsUbi methylation and PLsUbi-driven GUS mRNA accumulation. The mean percentage of methylated cytosines in lettuce was higher than that in Arabidopsis (16.6% and 4.8%, respectively) although the difference was not significant at the 5% level. The mean mRNA accumulation in lettuce was lower than that in Arabidopsis (2.4 and 6.9, respectively), and the difference was significant at the 5% level. In lettuce, the percentages of methylated cytosines in Line U-L2 (33.8%) and U-L3 (34.1%) were higher than those in Line U-L1 (2.4%) and U-L5 (2.3%), while GUS mRNA accumulation in Line U-L2 (2.1 or 1.5) and U-L3 (0.3) was lower than that in Line U-L1 (8.3 or 5.6) and U-L5 (3.6 or 3.0). These results suggest the inhibitory effects of methylation on PLsUbi-driven expression.

In this study, we observed several differences between lettuce and Arabidopsis. First, we found differential methylation rates between lettuce and Arabidopsis, although the reason for this difference is unclear. Second, we found that GUS mRNA accumulation was higher when driven by PLsUbi-TLsUbi than by P35S-Tnos in lettuce, but was lower when driven by PLsUbi-TLsUbi than by P35S-Tnos in Arabidopsis. The reason for this difference remains unclear, but one possibility is heterologous usage of the promoter, as supported by studies in other plant species. The maize UbiI polyubiquitin promoter has been reported to be less active than P35S in Gladiolus (Kamo et al., 1995). In addition, a polyubiquitin promoter isolated from Gladiolus, which has high activity in reintroduced Gladiolus transgenic plants, has been found to have less activity when introduced into rice, tobacco and various floral monocot plants (Joung and Kamo, 2006). The mechanisms underlying the different methylation rates between lettuce and Arabidopsis will be investigated in future studies.

In conclusion, we demonstrated that the PLsUbi-TLsUbi expression system, compared with the P35S-Tnos system, induces higher transcriptional enhancement of a reporter gene in lettuce, and also induces higher translational enhancement of reporter-gene expression in both lettuce and Arabidopsis. This expression system may be useful in diverse applications inducing artificially downregulating genes of interest via RNA-interference-mediated suppression in lettuce, as well as inducing sufficient yields of transgenic proteins in both lettuce and Arabidopsis. In addition, the ability of the PLsUbi-TLsUbi system to induce high transgene expression in heterologous circumstances, such as that in Arabidopsis, suggests that the system may be usable in various higher plant species.

Acknowledgments

The authors thank Dr. Akira Kanazawa (Hokkaido University) for helpful advice and valuable suggestions on DNA-methylation analysis, and Dr. Masaharu Kuroda (Central Region Agricultural Research Center, NARO) for providing the pZK3B vector. We are grateful to Satoshi Matsuo and Machiko Fukuda (NIVFS) for technical advice on qRT-PCR experiments. We are also grateful to Satomi Negoro, Yumika Kitamura, Kimi Sanami, and Aiko Morikawa (NIVFS) for skillful technical assistance.

Literature Cited

•  Bhattacharyya,  J.,  A. H.  Chowdhury,  S.  Ray,  J. K.  Jha,  S.  Das,  S.  Gayen,  A.  Chakraborty,  J.  Mitra,  M. K.  Maiti,  A.  Basu and  S. K.  Sen. 2012. Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants. Plant Cell Rep. 31: 271–279.
•  Binet,  M. N.,  M.  Lepetit,  J. H.  Weil and  L. H.  Tessier. 1991. Analysis of a sunflower polyubiquitin promoter by transient expression. Plant Sci. 79: 87–94.
•  Bourdon,  V.,  A.  Harvey and  D. M.  Lonsdale. 2001. Introns and their positions affect the translational activity of mRNA in plant cells. EMBO Rep. 2: 394–398.
•  Callis,  J.,  T.  Carpenter,  C. W.  Sun and  R. D.  Vierstra. 1995. Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia. Genetics 139: 921–939.
•  Callis,  J.,  J. A.  Raasch and  R. D.  Vierstra. 1990. Ubiquitin extension proteins of Arabidopsis thaliana—structure, localization, and expression of their promoters in transgenic tobacco. J. Biol. Chem. 265: 12486–12493.
•  Clough,  S. J. and  A. F.  Bent. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743.
•  Christensen,  A. H.,  R. A.  Sharrock and  P. H.  Quail. 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18: 675–689.
•  Garbarino,  J. E.,  T.  Oosumi and  W. R.  Belknap. 1995. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol. 109: 1371–1378.
•  Giannino,  D.,  C.  Nicolodi,  G.  Testone,  E.  Di Giacomo,  M. A.  Iannelli,  G.  Frugis and  D.  Mariotti. 2008. Pollen-mediated transgene flow in lettuce (Lactuca sativa L.). Plant Breeding 127: 308–314.
•  Goto,  F.,  T.  Yoshihara,  N.  Murata,  M.  Toyama,  T.  Masuda,  H.  Ikeda and  N.  Matsumoto. 2001. Molecular breeding of high iron content lettuce (Lactuca sativa L.) (5)—Environmental risk evaluation of the transgenic lettuce plants introduced ferritin gene. Denryoku Chuo Kenkyujo Abiko Kenkyujo Hokoku U01017: 1–22 (in Japanese).
•  Hajdukiewicz,  P.,  Z.  Svab and  P.  Maliga. 1994. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25: 989–994.
•  Hernandez-Garcia,  C. M.,  A. P.  Martinelli,  R. A.  Bouchard and  J. J.  Finer. 2009. A soybean (Glycine max) polyubiquitin promoter gives strong constitutive expression in transgenic soybean. Plant Cell Rep. 28: 837–849.
•  Hirai,  T.,  A. M.  Shohael,  Y. W.  Kim,  M.  Yano and  H.  Ezura. 2011. Ubiquitin promoter–terminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce. Plant Cell Rep. 30: 2255–2265.
•  Joung,  Y. H. and  K.  Kamo. 2006. Expression of a polyubiquitin promoter isolated from Gladiolus. Plant Cell Rep. 25: 1081–1088.
•  Kamo,  K.,  A.  Blowers,  F.  Smith,  J.  Van Eck and  R.  Lawson. 1995. Stable transformation of Gladiolus using suspension cells and callus. J. Amer. Soc. Hort. Sci. 120: 347–352.
•  Kamo,  K.,  A. Y.  Kim,  S. H.  Park and  Y. H.  Joung. 2012. The 5'UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis. BMC Plant Biol. 12: 79.
•  Kanazawa,  A.,  M.  O’Dell and  R. P.  Hellens. 2007. The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro. Plant Biol. (Stuttg) 9: 435–441.
•  Kawazu,  Y.,  R.  Fujiyama,  K.  Sugiyama and  T.  Sasaya. 2006. A transgenic lettuce line with resistance to both Lettuce big-vein associated virus and Mirafiori lettuce virus. J. Amer. Soc. Hort. Sci. 131: 760–763.
•  Kawazu,  Y.,  R.  Fujiyama,  Y.  Noguchi,  M.  Kubota,  H.  Ito and  H.  Fukuoka. 2010. Detailed characterization of Mirafiori lettuce virus-resistant transgenic lettuce. Transgenic Res. 19: 211–220.
•  Kawazu,  Y.,  R.  Fujiyama,  S.  Imanishi,  H.  Fukuoka,  H.  Yamaguchi and  S.  Matsumoto. 2016. Development of marker-free transgenic lettuce resistant to Mirafiori lettuce big-vein virus. Transgenic Res. 25: 711–719.
•  Kim,  D. H.,  Z. Y.  Xu and  I.  Hwang. 2013. AtHSP17.8 overexpression in transgenic lettuce gives rise to dehydration and salt stress resistance phenotypes through modulation of ABA-mediated signaling. Plant Cell Rep. 32: 1953–1963.
•  Kosugi,  S.,  Y.  Ohashi,  K.  Nakajima and  Y.  Arai. 1990. An improved assay for β-glucuronidase in transformed cells: Methanol almost completely suppresses a putative endogenous β-glucuronidase activity. Plant Sci. 70: 133–140.
•  Kuroda,  M.,  M.  Kimizu and  C.  Mikami. 2010. A simple set of plasmids for the production of transgenic plants. Biosci. Biotechnol. Biochem. 74: 2348–2351.
•  Lam,  E.,  P. N.  Benfey,  P. M.  Gilmartin,  R. X.  Fang and  N. H.  Chua. 1989. Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. Proc. Natl. Acad. Sci. USA 86: 7890–7894.
•  Lee,  K.,  S. M.  Lee,  S. R.  Park,  J.  Jung,  J. K.  Moon,  J. J.  Cheong and  M.  Kim. 2007. Overexpression of Arabidopsis homogentisate phytyltransferase or tocopherol cyclase elevates vitamin E content by increasing gamma-tocopherol level in lettuce (Lactuca sativa L.). Mol. Cells 24: 301–306.
•  Liu,  Y. G. and  R. F.  Whittier. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674–681.
•  McCabe,  M. S.,  F.  Schepers,  A.  van der Arend,  U.  Mohapatra,  A. M.  de Laat,  J. B.  Power and  M. R.  Davey. 1999. Increased stable inheritance of herbicide resistance in transgenic lettuce carrying a petE promoter-bar gene compared with a CaMV 35S-bar gene. Theor. Appl. Genet. 99: 587–592.
•  Mishiba,  K.,  M.  Nishihara,  T.  Nakatsuka,  Y.  Abe,  H.  Hirano,  T.  Yokoi,  A.  Kikuchi and  S.  Yamamura. 2005. Consistent transcriptional silencing of 35S-driven transgenes in gentian. Plant J. 44: 541–556.
•  Nagata,  R. T.,  J. A.  Dusky,  R. J.  Ferl,  A. C.  Torres and  D. J.  Cantliffe. 2000. Evaluation of glyphosate resistance in transgenic lettuce. J. Am. Soc. Hort. Sci. 125: 669–672.
•  Norris,  S. R.,  S. E.  Meyer and  J.  Callis. 1993. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol. Biol. 21: 895–906.
•  Okumura,  A.,  A.  Shimada,  S.  Yamasaki,  T.  Horino,  Y.  Iwata,  N.  Koizumi,  M.  Nishihara and  K.  Mishiba. 2016. CaMV-35S promoter sequence-specific DNA methylation in lettuce. Plant Cell Rep. 35: 43–51.
•  Rose,  A. B. 2004. The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant J. 40: 744–751.
•  Rose,  A. B. 2008. Intron-mediated regulation of gene expression. Curr. Top. Microbiol. Immunol. 326: 277–290.
•  Samadder,  P.,  E.  Sivamani,  J.  Lu,  X.  Li and  R.  Qu. 2008. Transcriptional and post-transcriptional enhancement of gene expression by the 5'-UTR intron of rice rubi3 gene in transgenic rice cells. Mol. Genet. Genomics 279: 429–439.
•  Siebert,  P. D.,  A.  Chenchik,  D. E.  Kellogg,  K. A.  Lukyanov and  S. A.  Lukyanov. 1995. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23: 1087–1088.
•  Sivamani,  E. and  R.  Qu. 2006. Expression enhancement of a rice polyubiquitin gene promoter. Plant Mol. Biol. 60: 225–239.
•  Sullivan,  J. A.,  K.  Shirasu and  X. W.  Deng. 2003. The diverse roles of ubiquitin and the 26S proteasome in the life of plants. Nat. Rev. Genet. 4: 948–958.
•  Sun,  H. J.,  M. L.  Cui,  B.  Ma and  H.  Ezura. 2006. Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce. FEBS Lett. 580: 620–626.
•  Sunilkumar,  G.,  L.  Mohr,  E.  Lopata-Finch,  C.  Emani and  K. S.  Rathore. 2002. Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol. Biol. 50: 463–474.
•  Wei,  H.,  M. L.  Wang,  P. H.  Moore and  H. H.  Albert. 2003. Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J. Plant Physiol. 160: 1241–1251.
•  Wickens,  M. and  P.  Stephenson. 1984. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation. Science 226: 1045–1051.