A Novel Moderate Constitutive Promoter Derived from Poplar (Populus tomentosa Carrière)

A novel sequence that functions as a promoter element for moderate constitutive expression of transgenes, designated as the PtMCP promoter, was isolated from the woody perennial Populus tomentosa. The PtMCP promoter was fused to the GUS reporter gene to characterize its expression pattern in different species. In stable Arabidopsis transformants, transcripts of the GUS reporter gene could be detected by RT-PCR in the root, stem, leaf, flower and silique. Further histochemical and fluorometric GUS activity assays demonstrated that the promoter could direct transgene expression in all tissues and organs, including roots, stems, rosette leaves, cauline leaves and flowers of seedlings and maturing plants. Its constitutive expression pattern was similar to that of the CaMV35S promoter, but the level of GUS activity was significantly lower than in CaMV35S promoter::GUS plants. We also characterized the promoter through transient expression in transgenic tobacco and observed similar expression patterns. Histochemical GUS staining and quantitative analysis detected GUS activity in all tissues and organs of tobacco, including roots, stems, leaves, flower buds and flowers, but GUS activity in PtMCP promoter::GUS plants was significantly lower than in CaMV35S promoter::GUS plants. Our results suggested that the PtMCP promoter from poplar is a constitutive promoter with moderate activity and that its function is presumably conserved in different species. Therefore, the PtMCP promoter may provide a practical choice to direct moderate level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.

Because of these potential risks, research on novel plant sequences that function as promoter elements for moderate constitutive level expression of transgenes is becoming increasingly significant.
The CaMV 35S promoter is a strong promoter; not only does it affect the associated transgene, it can also affect genes thousands of base pairs up-or down-stream of the insertion site on a given chromosome or even affect the behavior of genes on other chromosomes. In Brassica napus plants containing the 35S promoter from the mosaic virus (CaMV), it has been shown that upon infection with CaMV the driven transgene is silenced [46]. CaMV is a common pathogen of Brassica sp., and oilseed rape genetically modified to be resistant to herbicide using the CaMV promoter loses that resistance when it encounters naturally CaMV, indicating that transgene phenotypes can be modified by pathogen invasion [40]. The presence of two or more chimeric genes in the same transformation vector driven by the same constitutive promoter in a single plant may result in homology-dependent gene silencing, particularly where the promoter is also highly active [47], and can involve interactions between closely linked repetitive elements on one chromosome or homologous sequences on separate chromosomes [42,45]. In this case, the reason for gene silencing is the inactivation of the CaMV 35S promoter [38,43]. In cases that introduce another gene into transgenic plants, if the resident transgene contains the CaMV 35S promoter, the introduction of additional copies of the 35S enhancer may result in methylation and silencing of unlinked homologous copies [41,44]. Thus, finding novel promoters for moderate constitutive level expression of transgene is becoming increasingly useful.
Poplar is a woody perennial that has been the subject of many studies focused on tissue-specific and inducible promoters [62][63][64][65][66][67][68], but relatively few studies about poplar constitutive promoters have been reported. There is a demand for moderate constitutive promoters; for instance, a very strong promoter would not be appropriate when expressing transgenes that are toxic or otherwise inhibitory in abundance [69]. Here, we cloned and analyzed a novel sequence from P. tomentosa that functions as a promoter element for moderate constitutive expression of transgenes, designated as the PtMCP promoter. A series of functional characterizations of the PtMCP promoter in stable Arabidopsis transformants were performed using RT-PCR, histochemical staining and fluorometric assays of GUS activity. We also report the expression patterns and quantitative measurements of GUS activity of the PtMCP promoter transiently expressed in tobacco.

Construction and Transformation of the PtMCP Promoter::GUS Construct into Arabidopsis
To investigate the expression pattern of the PtMCP promoter, the isolated promoter fragment was fused with the GUS reporter gene in the pProtest vector to generate a PtMCP promoter::GUS construct ( Figure 2). The resultant plasmid was stably introduced into Arabidopsis by the floral dip method. The binary vector pBI121, in which the GUS gene was driven by the cauliflower mosaic virus (CaMV) 35S promoter ( Figure 2B), was transformed as the positive control, and wild-type Arabidopsis was used as a negative control. Consequently, we obtained 10 independent transgenic lines for PtMCP promoter::GUS. Of these, eight independent transgenic lines were used for further analysis.

Characterization and Analysis of Transgenic Arabidopsis
The stable integration of T-DNA into the genome of the transgenic plants was verified by genomic PCR analysis using GUS gene-specific primers. An expected 239 bp specific amplification product for the GUS gene was obtained from all eight transgenic lines tested (T2-T5, T7-T9, T11), and no amplification signal was observed in the wild-type control plants ( Figure 3A). Furthermore, RT-PCR using in vitro grown plants demonstrated that the transcript of the GUS reporter gene was present in the eight transgenic lines; the GUS transcript was not detected in wild-type control plants. Notably, the GUS expression levels varied among different transgenic lines, and RT-PCR revealed that the transcript abundance of GUS in all transgenic lines was lower than that of the reference ACTIN2 gene. The GUS transcript abundance of the T7 transgenic line was the highest ( Figure 3B). To determine the tissue-specificity of the PtMCP promoter in Arabidopsis, RT-PCR was performed. RT-PCR indicated that the GUS reporter gene was transcribed in the root, stem, leaf, flower and silique of Arabidopsis. The GUS transcript abundance was similar among the various tissues and organs and was lower than that of the reference ACTIN2 gene ( Figure 3C). WT, wild-type plants serving as negative controls; P, positive control (PtMCP promoter::GUS plasmid). The Arabidopsis ACTIN2 gene was used as an endogenous reference gene for the RT-PCR assay and also amplified to verify that similar amounts of cDNA were used. The RT-PCR data are representative of at least three experiments. Numbers on the left represent the size of the amplified cDNA fragments in base pairs.

Histochemical and Fluorometric GUS Assays
To understand the spatial expression pattern of the PtMCP promoter, histochemical GUS activity was analyzed in different tissues and organs of transgenic Arabidopsis seedlings and maturing plants (T2, T7) in the absence of environmental influences. Histochemical staining showed moderate GUS activity in the roots, stems and leaves of seedlings and the roots, stems, rosette leaves, cauline leaves and flowers of maturing plants, but it was weaker than in the corresponding tissues of positive controls (CaMV35S promoter::GUS). There was no GUS activity observed in these tissues in the negative controls (wild-type plants) ( Figure 4A-H). Quantitative measurement of GUS activity also showed that GUS activity in the PtMCP promoter::GUS plants (T2, T4, T7, T8, T9 and T11) was significantly lower than in the CaMV35S promoter::GUS plants ( Figure 4I). These results indicated that the PtMCP promoter was constitutive, but its expression intensity was significantly lower than the CaMV35S promoter.  Figure 5J) and flower ( Figure 5P), but it was weaker than the GUS activity in the corresponding tissues and organs of positive controls (CaMV35S promoter::GUS) ( Figure 5B,E,H,K,N,Q). No visible GUS activity was detected in these tissues and organs in wild-type plants ( Figure 5C,F,I,L,O,R). To increase the sensitivity of GUS reporter gene detection, quantitative measurements were made ( Figure 6). Moderate GUS activity was detected in the root, stem, leaf and flower, but the protein levels differed in the various tissues. The maximum amount of GUS protein was detected in the leaf, while levels were lowest in the stem ( Figure 6A). GUS activity in the PtMCP promoter::GUS plants was significantly lower (p < 0.01) than in corresponding tissues of CaMV35S promoter::GUS plants. GUS activity in the negative controls (wild-type plants) was negligible ( Figure 6B). These results were identical with the results in Arabidopsis ( Figure 4); the PtMCP promoter was moderately constitutive. Moreover, a similar expression pattern was also observed in poplar (data not shown).

Discussion
To investigate the expression mechanism of the PtMCP promoter, a 2661 bp sequence was isolated and analyzed in this study. The spatial and temporal expression patterns of this promoter were first clarified by assaying GUS activity in transgenic Arabidopsis plants containing a PtMCP promoter::GUS fusion construct. The construct was stably transformed into Arabidopsis and transiently introduced into tobacco to examine whether the function of this promoter is conserved between different species.
Using the PLACE [71,72] and PlantCARE [73] databases, putative TATA and CAAT-boxes were located at positions 19 and 74 relative to the transcription start site "A", and several other common cis-elements were also identified in the PtMCP promoter. The G-box motif is a light responsive cis-acting element that has been found to be essential for transcriptional activity in the Arabidopsis rbcS-1A promoter [74], the spinach ribulose bisphosphate carboxylase/oxygenase (RuBisCO) activase promoter [75] and the rice OsActin2 gene regulatory region [25]. TC-rich repeats are involved in defense and stress responses and are also found in yeast [76] and barley [77] ubiquitin extension protein promoters. The TATC-box and the GARE-motif are involved in gibberellin-responsiveness, BoxI, Box4, the GAG-motif, Sp1 and the TCT-motif are light responsive elements, MBS is an MYB binding site involved in drought-inducibility, TCA-elements are involved in salicylic acid responsiveness, the CAT-box is related to meristem expression, LTR is involved in low-temperature responsiveness, Box-W1 is a fungal elicitor responsive element and ABRE is involved in abscisic acid responsiveness.
In transgenic Arabidopsis, histochemical GUS staining showed GUS activity in all tissues and organs (roots, stems, rosette leaves, cauline leaves and flowers) of seedlings and maturing plants, but it was weaker than the GUS activity in CaMV35S promoter::GUS plants. Furthermore, quantitative measurement of GUS activity also proved that GUS activity in the PtMCP promoter::GUS plants was significantly lower than in CaMV35S promoter::GUS plants. These results were in agreement with the RT-PCR results; RT-PCR indicated that the GUS reporter gene was transcribed in the root, stem, leaf, flower and silique of Arabidopsis. The reason why different GUS expression levels among transgenic Arabidopsis lines may be the T-DNA insertion position in plant genome and different copy number.
To contrast with the expression pattern in Arabidopsis, the same PtMCP promoter::GUS construct was transformed into tobacco transiently, and a similar expression pattern was observed. Histochemical GUS staining showed GUS activity in all tissues and organs of tobacco, including the root, stem, leaf, flower bud and flower, but it was weaker than the GUS activity in CaMV35S promoter::GUS plants. Quantitative analyses showed that GUS activity in the PtMCP promoter::GUS plants was significantly lower (p < 0.01) than in the corresponding tissues and organs of CaMV35S promoter::GUS plants.

Plant Materials and Growth Conditions
The poplar (Populus tomentosa Carrière) clone TC1521 was used as source material for this study. Poplar plants were grown in a growth chamber under long-day conditions (16-h light/8-h dark, cool white fluorescent light, minimum illumination: 0.2 mM/s·m 2 ) at 22 °C. Four-month-old plants with an identical growth status were then used for young leaf collection and prepared for cloning of the PtMCP promoter.
Plants of Arabidopsis thaliana L. ecotype Columbia (Col) used in plant transformation were grown and maintained in the greenhouse of the National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing. Arabidopsis seeds were sterilized and placed on 1/2 Murashige-Skoog (MS) solid medium [78] at 4 °C for 2-4 days. The seedlings were grown in a growth chamber under the same conditions as poplar plants for 10 day before being transplanted to artificial soil mix composed of humus, vermiculite and perlite [1:2: Tissue-cultured tobacco plants (Nicotiana tabacum cv. W38) were raised and synchronized (using vegetative stem cuttings containing an axillary bud) on 1/2 Murashige-Skoog (MS) solid medium [78] supplemented with 0.4 mg/L IBA and adjusted to pH 5.8. In vitro and tissue-cultured tobacco plants were grown in a growth chamber under the same conditions as poplar plants before being used for Agrobacterium-mediated transient expression assays.

Bacterial Strains
Escherichia coli (strain DH5α) was used for the cloning and propagation of all recombinant plasmid vectors. Agrobacterium tumefaciens (strain GV3101) was used to transform A. thaliana (Col) plants via the floral dip method [79,80]. The plasmids pProtest (Obtained from the Laboratory of Professor Steven H. Strauss, Oregon State University, Corvallis, OR, USA), pMD19-T vector and pBI121 (Clontech, Mountain View, CA, USA) were used to generate promoter fragment constructs.

Construction of the PtMCP Promoter::GUS Plasmid
To construct a binary vector consisting of the GUS coding sequence driven by the PtMCP promoter, a fragment of approximately 2.6 kb was obtained by PCR amplification using P. tomentosa genomic DNA as a template. Genomic DNA was extracted from leaves (300 mg) using a modified CTAB extraction method, as previously described [81]. Primers with additional restriction sites were designed: The forward primer 5'-CTACGAGCTCTACTAAATAAATATATAA-3' and the reverse primer 5'-ATGGTACCATCTATCTGCCCCCTTGTC-3′. The restriction enzyme sites SacI (in the forward primer) and KpnI (in the reverse primer) are underlined. PCR reactions were carried out in 20 μL volumes under the following conditions: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 57 °C for 30 s and 68 °C for 2.5 min. PCR products were subcloned into the pMD19-T vector (TaKaRa, Otsu, Japan) and then sequenced. The promoter fragment was analyzed using the PLACE [71,72] and PlantCARE [73] databases to find putative functional promoter elements. The PtMCP promoter-T construct was digested with SacI/KpnI and then cloned into the corresponding site of the expression vector pProtest. The resultant PtMCP promoter::GUS construct was transformed into A. tumefaciens strain GV3101 by electroporation [82].

Arabidopsis Transformation
A. tumefaciens strain GV 3101 containing the PtMCP promoter::GUS construct was used to transform A. thaliana (Col) plants via the floral dip method [79,80]. Kanamycin (50 μg/mL) resistant transformants (T0) were transplanted from the proliferating medium to artificial soil mix and grown in a growth chamber set at 22 °C under LD conditions. Further analyses were performed, too.

Genomic PCR and RT-PCR Assays
For preliminary verification of the presence of transgenes, putative transgenic plants were screened by PCR. Total genomic DNA was extracted from all of the kanamycin-resistant plants with a Plant Genomic DNA Kit (TIANGEN, Beijing, China). PCR was performed in a total volume of 20 μL, with 2 μL of 10× PCR buffer, 1.6 μL of 2.5 mM dNTPs, 2 μL plant genomic DNA(100 ng/L) 0.4 μL each of forward and reverse primers (10 μM), 0.2 μL Taq DNA polymerase (5000 U/mL) and 13.4 μL ddH 2 O. Thermal cycling was performed at 94 °C for 5 min, then 94 °C for 30 s, 61 °C for 15 s and 72 °C for 20 s, for 30 cycles, with final extension at 72 °C for 5 min and, finally, kept at 4 °C, using the GeneAmp ® PCR System 9700 (ABI, Hong Kong, China). Genomic PCR was carried out using GUS gene-specific primers: forward primer 5'-GTTACGTCCTGTAGAAACCCCAACC-3' and reverse primer 5'-CTGCCCAACCTTTC GGTATAAAGAC-3'.
Total RNA was extracted from Arabidopsis samples, according to the method described previously [83]. Total RNA was pre-treated with RQ1 DNase I (Promega, Madison, WI, USA) to eliminate residual genomic DNA. First-strand cDNA was synthesized using 1.0 μg of DNase-treated total RNA, Superscript III (Invitrogen, Carlsbad, CA, USA) and oligo d(T) 20 in a total volume of 20 μL. The first-strand cDNA was diluted 1:10 with ddH 2 O, and 2 μL of the diluted cDNA was used as a template for RT-PCR analysis. RT-PCR was performed in a total volume of 20 μL. GUS primers: forward primer 5'-GTTACGTCCTGTAGAAACCCCAACC-3' and reverse primer 5'-CTGCCCAACC TTTCGGTATAAAGAC-3'. The A. thaliana ACTIN2 gene (Phytozome v8.0 accession: AT3G18780) was used as an endogenous control gene with the primers: forward 5′-AAGCACA ATCCAAGAGAGGTATTC-3' and reverse 5'-TACATAGCGGGAGAGTTAAAGGTC-3'. To detect expression patterns and transcript levels of the PtMCP promoter in Arabidopsis, total RNA was extracted from various tissues (root, stem, leaf, flower, silique). The expression pattern of PtMCP promoter in Arabidopsis was then analyzed by RT-PCR. PCR products were evaluated following agarose gel electrophoresis and spectrophotometrical analysis.
Fluorometric assay for GUS activity was performed according to the method of Jefferson [84]. The transgenic plants were ground in liquid nitrogen and homogenized in freshly prepared GUS extraction buffer (50 mM phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% (w/v) sodium lauryl sarcosine, 10 mM β-mercaptoethanol). The homogenate was then centrifuged for 10 min at 12,000 rpm at 4 °C, and the GUS activity of the supernatant was assessed using 4-methylumbelliferyl glucuronide (4-MUG) as a substrate. The fluorescence of the GUS-catalyzed hydrolysis reaction product, 4-methylumbelliferone (4-MU), was measured with the TECAN GENios system (Tecan, Shanghai, China). The protein concentration in the supernatant was determined by the procedure of Bradford [85], using bovine serum albumin (BSA) as a standard. GUS activity was normalized to the protein concentration of each supernatant extract and expressed as picomoles 4-MU per minute per milligram protein.

Agrobacterium-Mediated Transient Expression in Tobacco
For Agrobacterium-mediated transient expression of the PtMCP promoter in tobacco, root, stem, leaf, flower bud and flower tissues were infiltrated into Agrobacterium culture (OD 600 = 0.8). After 20-30 min of vacuum filtration, the infiltrated samples were blotted dry with sterile filter paper and maintained in petri-dishes containing co-cultivation medium (MS medium) at 25 °C in a dark place for 48 h. The samples were then collected and subjected to histochemical and fluorometric GUS assays.

Conclusions
A novel constitutive promoter with moderate activity from P. tomentosa, designated the PtMCP promoter, has one TATA-box, one CAAT-box and several other putative cis-acting regulatory elements. The expression mechanism of the PtMCP promoter is similar to that of the CaMV 35S promoter, but the levels of activity are considerably lower. The function of the PtMCP promoter from poplar is presumably conserved in different species. Therefore, the PtMCP promoter described in this paper provides a practical choice to direct moderate level constitutive expression of transgenes. The emergence of this promoter could perhaps provide a valuable new tool for scientists in plant genetic engineering.