Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

Background Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. Methodology/Principal Findings We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. Conclusion and Significance We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells.


Introduction
The overall strength of a promoter as well as its tissue-specific expression pattern depends on the combination of the spatial orientation of cis-elements and interaction with nuclear protein factors [1,2]. Such interactions suggest that the cis-elementcontaining upstream activation sequence (UAS) plays an important role in determining promoter function [3]. Moving the DNA element (cis-factor) that binds a specific trans-factor from one promoter into a different promoter can result in a novel transcription model [4,5], that can modulate the transcriptional activity and cell specific expression pattern of the promoter [6]. Several recombinant/synthetic promoters have been created earlier by (a) ligating the upstream activation sequence (UAS) of one promoter with the TATA box containing domain of another promoter; like tacI/tacII hybrid promoters [7], E4/E8 hybrid promoters [8] and chimeric plant promoter (Mac promoter) [9]; (b) placing desired cis-elements in conjunction with heterologous promoters [10]; (c) bringing together cis-elements from different promoters [11][12][13]. The strength and tissue specificity of chimeric promoters derived by incorporating sub-domains of the mannopine synthase (mas2) and octopine synthase (ocs) promoters were investigated earlier [14]. The Mac promoter, incorporating the mas region from +65 to 2301 and the CaMV35S enhancer region from 290 to 2941, has been reported to enhance the level of GUS expression by several folds as compared to the CaMV35S promoter [9]. Similarly, the strength and tissue specificity of Mac-and super-promoters were analyzed [14][15][16]. Novel plant transformation vectors incorporating the super-promoter were tested in transgenic tobacco and maize plants, and also transiently in maize protoplasts [16].
Risk of successive transformation by single promoter is that it might lead to silencing of transgenes in successive generations by homologous recombination [13,[17][18][19][20]. Use of different heterologous promoters is necessary during pyramiding (stacking) of genes in a single plant cell for developing a particular trait in plants. As in the case of developing b carotene (Provitamin A) enriched rice multiple genes coupled to different promoters were inserted in rice endosperm for engineering the concerned metabolic pathway/s [21].
In the present study, we have characterized a set of 10 unique pararetrovirus-based recombinant sub-genomic transcript promoters, and their expression activities were compared to that of the CaMV35S, MS8 and FS3 promoters in transient expression systems. An in-depth expression analysis of one of these hybrid promoters (MSgt-FSgt), which conferred the highest level of activity, was further studied in transgenic tobacco and Arabidopsis plants. Correlation between the MSgt-FSgt promoter-driven GUS activity and the uidA-mRNA level in transgenic plants was assayed. The interaction between the MSgt-FSgt promoter sequence and tobacco nuclear proteins was also analyzed by Electrophoretic Mobility Shift Assay (EMSA) and DNA Footprinting. The cell specificity of MSgt-FSgt was compared to that of the CaMV35S promoter in different plant cells of the stem, leaf, and root using Confocal Laser Scanning Microscopy (CLSM). The recombinant MSgt-FSgt promoter reported here will be a valuable addition to the tools available for plant gene expression studies.

Materials
Restriction and modifying enzymes were purchased from Promega (Madison, WI USA), and were used according to the manufacturer's instructions. The Nytran membrane was obtained from Schleicher & Schuell (Keene, NH, USA). All fine chemicals, including MUG, X-gal, X-gluc, DEPC were purchased from Sigma-Aldrich (St. Louis, USA). Platinum high fidelity Taq DNA polymerase was obtained from Gibco-BRL (Maryland, USA). DNA methyltransferase M.SssI and E.coli K12 ER2925 were purchased from New England Biolabs, Beverly, MA, USA.

Promoter-GFP constructs for transient assay
The GFP cDNA gene was PCR-amplified using a synthetic primer pair (Table 2) to insert the XhoI site at the 59-end and the SstI site at the 39-end. The amplified GFP gene was digested with XhoI and SstI and subsequently cloned into the XhoI and SstI sites of pUCPMAGUS, pFS3GUS, pMS8GUS and pUPMSgt-FSgtGUS (set-a) replacing the GUS gene to generate plasmids pUCPMAGFP, pFS3GFP, pMS8GFP and pUPMSgt-FSgtGFP (set-b) respectively.

Construction of plant expression vectors
The MSgt-FSgt promoter fragment 369-bp from pUPMSgt-FSgt was gel purified (as EcoRI -HindIII fragment) and inserted into pKYLXGUS, a plant expression vector [26,27] using EcoRI and HindIII sites that flank the promoter, thereby replacing the CaMV35S promoter. The resulting plant expression vector was designated as pKMSgt-FSgtGUS.

Protoplast isolation, electroporation and transient assay of recombinant promoters
Protoplasts from a tobacco cell suspension culture (Nicotiana tabacum cv. Xanthi Brad) were purified on a 20% sucrose gradient after digestion by cellulase (Sigma, USA) and pectinase (Sigma, USA) and electroporated following a standard protocol as    poration cuvette (0.4-cm electrode gap). The plasmid pUCPMA-GUS containing the CaMV35S promoter [26] was used as a control to compare the activities of the promoters. GUS activities in transformed protoplasts were measured after 20 h of incubation at 28uC [28]. The average activities of these promoter constructs were expressed as the mean of five successive independent experiments.

Promoter expression analysis using CLSM
Tobacco protoplasts were electroporated with promoter constructs carrying GUS reporter (set a) and GFP reporter (set b) constructs for assaying their transient activities as described earlier.
Protoplasts electroporated with promoter constructs carrying the GUS reporter (set-a) were incubated in a 1mM MUG (4-methylumbelliferyl-beta-d-glucuronide) at 37uC for 30 in to generate 4-MU (7-hydroxy-4-methylcoumarin) for the detection of GUS localization (as blue fluorescence) in protoplasts using CLSM (TCS SP5, Leica Microsystems CMS GmbH, D-68165 Mannheim, Germany) with similar specifications as described earlier [29]. Similarly, to excite the expressed GFP [30,31] in protoplasts, the argon laser (40%) with AOTF of 488 nm (at 30%) was used. The fluorescence emissions were collected between 501 and 598 nm as described earlier [29]. Following image acquisition, 4-MU (for GUS) and GFP fluorescence intensities were quantified using the LAS AF Software as per the instructions of Leica Microsystems. The GFP and GUS (4-MU) fluorescence intensities from 200 individual protoplasts were assayed, and the mean data were presented with respective 6 SD.

Transformation of Arabidopsis thaliana plants and analysis of transgenic plants
Arabidopsis thaliana (ecotype Columbia) plants were transformed by pKYLXGUS and pKMSgt-FSgtGUS by floral dip method [35]. The Arabidopsis flower buds were dipped in an Agrobacterium cell suspension containing freshly prepared 5% sucrose (wt/ vol) and 0.02-0.05% Silwet L-77 (vol/vol) for 30-45 sec. After that these plants were grown at 22uC under long day condition (16 hours light and 8 hours dark cycle) till setting of seeds. Seeds were collected after maturation and dried. After surface sterilization, seeds were suspended in sterile 0.05% agarose and spreaded on MS selection plate (4.3 g Murashige & Skoog salts, 10 g sucrose, 0.5 g MES, 8 g agar per liter; pH 5.7, Kanamycin 100 mg/l and Cefotaxime 100 mg/l) and allowed to germinate. Only true transformants produced green healthy leaves (nontransformants became dried and bleached). A total number of 75 independent transgenic plants were raised. GUS activities in whole seedlings (21 days old) were measured according to the protocol described earlier [28]. Histochemical GUS staining was performed as described earlier.

Molecular analysis of transgenic plants: RNA isolation, northern blot analysis
Total RNA was obtained from transgenic seedlings expressing pKYLX, pKYLXGUS, pKMS8GUS, pKFS3GUS and pKMSgt-FSgtGUS using the RNeasy plant mini Kit (Qiagen, Tokyo, Japan) according to manufacturer's protocol. The GUS and b-Actin genes were PCR-amplified from pKYLXGUS plasmid (for GUS) and tobacco genomic DNA [36] respectively. The PCR fragments were gel-purified using the QIAquick gel extraction kit (Qiagen, Tokyo, Japan) and was labeled with [a-P 32 ] dCTP using the Random Primer labeling Kit (NEBlot Kit, New England Biolabs Inc., MA, USA). Total RNA (5 mg) isolated from the transgenic seedlings was subjected to electrophoresis in a 1.2% agarose gel containing 0.66 M formaldehyde and then transferred overnight to an IMMOBILON-NY+ membrane (Millipore, Billerica, MA, USA) by capillary action in 10X SSC (1.5M NaCl, 150 mM sodium citrate). Hybridization with the 32 P labeled GUS-probe was carried out at 65C overnight and northern blot analysis was carried out using standard protocol [37]. The washed membrane with the bound probe was scanned with the FLA-5000 Imaging System (Fuji FILM Life Science, USA). The membrane was subsequently washed at 80uC in a solution containing 1X SSC, 0.1% SDS, and 0.05% Na-pyrophosphate to remove the GUS probe. The stripped membrane was then re-probed with the b-Actin to ensure equal loading.

Real-time PCR analysis of GUS transcripts driven by MS8, FS3, CaMV35S and MSgt-FSgt promoter
Total RNA was isolated from transgenic seedlings expressing the pKMS8GUS, pKFS3GUS, pKYLXGUS, and pKMSgt-FSgtGUS promoter constructs and subsequently mRNA was isolated from total RNA using the PolyATract mRNA isolation system (Promega, Madison, WI, USA). About 2.0 mg of mRNA was used to synthesize the first strand cDNA using AMV reverse transcriptase and oligo (dT) primers at 42uC for 1.5 hours. Real time PCR reactions were performed using SYBR Premix Ex TaqTM II (Perfect Real Time, Takara Bio Inc.) according to manufacturer's instruction on an Opticon-2 qRT-PCR instrument (MJ Research, Bio-Rad; Model CFD-3220). GAPDH cDNA was used as an internal control for normalization of GUS mRNA levels in the real time PCR. The PCR cycling program was conducted as follows; 94uC for 30 sec followed by 37 cycles of 94uC for 5 sec, 57uC for 30 sec, 72uC for 30 sec and finally 72uC for 5 minutes. The GUS mRNA levels in MS8, FS3 and MSgt-FSgt and CaMV35S promoters were calculated using the 2 2DDCT method [38,39] and data presented here are relative to CaMV35S promoter line.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
First strand cDNA was synthesized using 2.0 mg mRNA purified from total RNA extracted from transgenic tobacco seedlings transformed with pKYLX, pKYLXGUS, pKMS8GUS, pKFS3GUS and pKMSgt-FSgtGUS and transgenic Arabidopsis seedlings transformed with pKYLX, pKYLXGUS and pKMSgt-FSgtGUS as described above. PCR amplifications of GUS and GAPDH were performed using 1 ml of first strand reaction products in the presence of gene specific primers ( Table 2). Twenty-six cycles of PCR was conducted with denaturation at 92uC for 1 minute, annealing at 60uC for 30 seconds and extension at 72uC for 1 minute. Eight ml samples of each PCR reaction were analyzed on 1% agarose gel.

Preparation of transgenic plant samples for CLSM analysis
Transgenic tobacco plants were generated using the MSgt-FSgt and CaMV35S promoters fused to the GUS reporter gene as described earlier. The transgenic plant parts were initially kept under vacuum infiltration for 10 min and then incubated at 37uC overnight in the presence of 1 mM MUG to produce 4-MU. The transverse sections (approximately 80 micron thick) of leaf blade, leaf midrib, stem and root were obtained by sectioning the treated plant tissue using a microtome (Cryostat, Leica CM 1850). Fluorescence images of thin transverse sections of these tissues were captured using a CLSM. To quantify GUS activity, the treated tissue sections were excited with the 405 diode laser (Argon 364 nm UV laser may be more appropriate), and fluorescence emissions were collected between 440 and 455 nm with the detector (PMT) gain set at 1150V. Intracellular GUS localizations were detected by blue fluorescence of 4-MU using CLSM and intensities of blue-fluorescence were measured from different cells according to the protocol described earlier [29].
Nuclear protein binding assay for the hybrid promoter Tobacco nuclear extracts were prepared from greenhousegrown leaves using standard protocol [40] with slight modifica- tions. The MSgt-FSgt promoter DNA fragment (369 bp) was labeled using Prime-a-gene labeling system (Promega, USA) in the presence of c-P 32 dATP at 37uC for 1 hr. The EMSA binding reaction was carried out in a 30-ml volume containing 32 P-labeled MSgt-FSgt probe in binding buffer (100 mM Tris-HCl pH 7.5, 0.5 mM DTT, 1 mM EDTA, 7% v/v glycerol, 1 mM PMSF, 5 mg/ml BSA, 0.1-0.2 mg/ml poly dI-dC, 40-50 mM NaCl) in the presence of nuclear protein extract (5-6 mg). Reactions were incubated at room temperature for 30 min. The DNA-protein complexes were resolved in nondenaturing 5% polyacrylamide gel [40]. Competitive EMSA reactions were carried out in the presence of the 32 P-labeled MSgt-FSgt DNA (as probe) and 20, 50 and 100 fold excesses of non-labeled DNA (MSgt-FSgt).
DNaseI foot-printing experiments were carried using standard protocol [41]. The probe (end-labeled MSgt-FSgt promoter) was incubated in the presence of 100 mM Tris-HCl pH 7.5, 0.5 mM DTT, 1 mM EDTA, 7% v/v glycerol, 1 mM PMSF, 5 mg/ml BSA and 0.1-0.2 mg/ml polydI-dC for 30 min at room temperature. DNaseI (0.025 U/ml) was added and incubated for 45 sec at room temperature with 5 mg and 10 mg of nuclear protein separately. The reaction products were then denatured and loaded onto a 6% denaturing polyacrylamide sequencing gel along with sequencing reactions for the MSgt-FSgt DNA

In vitro methylation assay
The putative CG methylation sensitive sites present in MSgt-FSgt and the CaMV35S promoters were determined in-silico using Support Vector Machine (http://bio.dfci.harvard.edu/Methylator) [42]. The effect of in vitro methylation on the transient expression of MSgt-FSgt and CaMV35S promoters coupled to GUS reporter gene were investigated according to Pradhan et al. [43] with some modifications. Plasmids pUPMSgt-FSgtGUS and pUCPMAGUS individually were grown in a dam 2 and dcm 2 E.coli K12 strain ER2925 and purified. DNA methyltransferase M.SssI was used to methylate each plasmid construct at 37uC for 1.5 hours in presence of 0.5 mM S-adenosyl methionine. The methylated plasmids were purified and quantified using a spectrophotometer (CECIL BioQuest CE 2501 CECIL Instruments Ltd, England). Approximately 5 mg each methylated and unmethylated plasmids from both constructs were used for transient assay in a protoplast system (Nicotiana tabaccum cv. Xanthi brad) following protocol as described earlier.

Statistical Analysis
Statistical analyses were performed by using Graph Pad Prism (version 5.01). Unpaired students t test was used for analyzing the activity of methylated-MSgt-FSgt and methylated-CaMV35S promoters. A P value of ,0.05 was considered to reveal a significant difference.

Comparison of promoter activities of MS8, FS3, CaMV35S and MSgt-FSgt fused to GUS and GFP reporters using CLSM
Tobacco protoplasts were transformed with two different sets of promoter constructs as set-a and set-b separately as described in methods. In both cases the pUCPMA vector was used as a control. Blue fluorescence of transformed protoplasts obtained from using set-a (with GUS reporter gene; Figure 2a) were captured by CLSM

Analysis of transgenic tobacco plants developed with MS8, FS3, CaMV35S and MSgt-FSgt promoter constructs
Transgenic tobacco plants were generated using the promoter constructs pKYLX (empty vector), pKYLXGUS, pKMS8GUS, pKFS3GUS, pKMSgt-FSgtGUS. No phenotypic changes were marked between control and transgenic tobacco plants expressing these promoter constructs. Total proteins isolated from T 1 seedlings were used to assess GUS activity [28]. A comparison of the GUS activities of the various constructs with the CaMV35S (considered as 1) was presented in Figure 3a. In transgenic plants, the MSgt-FSgt promoter was found to exhibit 14.22, 4.26 and 2.94 times stronger activity, compared to the CaMV35S, MS8, and FS3 promoters, respectively (Figure 3a). Histochemical staining of transgenic tobacco seedlings (T 1 generation, 21 days old) generated with the CaMV35S, MS8, FS3 and MSgt-FSgt promoter constructs were presented in Figure 3b. When GUS activities were measured from soluble proteins extracted from the roots, leaves and stems of transgenic seedlings, the relative GUS activities in the tissues were in the following order: roots. leaves. stems (data not shown).
Northern blot analysis was carried out using total RNA isolated from transgenic seedlings generated with pKYLX, pKYLXGUS, pKMS8GUS, pKFS3GUS, pKMSgt-FSgtGUS. Equal loading of RNA for the northern blot analysis was confirmed (Figure 3c) and the results were shown in Figure 3d. The highest intensities of GUS transcript accumulation were observed for the MSgt-FSgt promoter, followed by the FS3, MS8 and CaMV35S promoters. The membrane was washed and reprobed using the b-Actin probe as discussed in the Methods for further confirmation of equal loading as shown in Figure 3e.
Accumulation of GUS-specific mRNAs driven by each promoter in transgenic tobacco plants (T 1 generation) were determined by semi-quantitative RT-PCR and real time PCR. The RTdependent PCR amplifications of GUS transcript driven by the CaMV35S, MS8, FS3 and MSgt-FSgt promoters in transgenic plants were shown in Figure 3f. Accumulation of GUS transcripts was found to be the highest in transgenic plants carrying the MSgt-FSgt promoter followed by the FS3, MS8 and CaMV35S promoters. The fold differences in the uidA-mRNA expression levels for CaMV35S, MS8, FS3 and MSgt-FSgt promoter constructs were measured by real-time PCR. Results were presented as the mean of three independent experiments with respective standard deviation (6 SD) in Figure 3i.

Comparison of MSgt-FSgt and CaMV35S promoter activity in transgenic Arabidopsis plant
We have generated approximately 75 independent transgenic Arabidopsis plants expressing following promoter constructs pKYLX (empty vector), pKYLXGUS, pKMSgt-FSgtGUS individually as described in Methods. Furthermore, transgenic Arabidopsis plants expressing MSgt-FSgt promoter were found to be phonotypically identical with wild type plants.
Histochemical staining of transgenic Arabidopsis seedlings expressing MSgt-FSgtGUS and CaMV35SGUS constructs were shown in Figure 4a; and the corresponding GUS activities in Figure 4b. The GUS activity of MSgt-FSgt was 7.16 times stronger than the CaMV35S promoter activity (Figure 4b). In the transgenic Arabidopsis plant, the RT-dependent PCR (semi q-RT PCR) amplification of GUS transcripts driven by the CaMV35S and MSgt-FSgt promoters were determined and it was observed that accumulation of GUS transcripts was more in transgenic Arabidopsis plants harboring the MSgt-FSgt promoter compared to that in transgenic plants with the CaMV35S promoter (Figure 4c).
The average GUS activity of 75 independent lines (21 days old) from each construct was evaluated from two independent experiments and the results were presented in Figure 5. Approximately 10 out of 75 (13.3%) independent transgenic Arabidopsis lines expressing CaMV35S showed the basal or minimum level of GUS expression while in case of MSgt-FSgt promoter 5 lines out of 75 (6.7%) transgenic Arabidopsis lines showed the basal or minimum level of GUS expression (Figure 5a, 5b and 5c).

Cell/Tissue specificity of MSgt-FSgt and CaMV35S promoter
The GUS gene expression levels in different cells/tissue types in the leaf, stem and root of tobacco plants transformed with the CaMV35S and MSgt-FSgt promoters were assayed using CLSM as described in the Method section. In the leaf blade, the MSgt-FSgt promoter exhibited 8.5760.93, 3.2960. 50, 7.5761.41, 6.4860.92, 90.269.95 and 20.0662.77 fold higher activity than the 35S promoter in the trichome, lower epidermis, spongy mesophyll, palisade mesophyll, subsidiary and guard cells, respectively (Figure 6a, 6b and 7a and Table 3). The expression acts of the MSgt-FSgt and CaMV35S promoters were also compared in the transgenic leaf midribs using CLSM-based techniques (Figure 6c and 7b). When compared, GUS gene expression under control of the hybrid promoter was found to be 7. 55 Figure 6d and 7c). In the case of root, the activity of the CaMV35S promoter was undetectable in root epidermal cells, moderate in root cortical cells and in phloem tissue, but exhibited the highest activity in the root xylem tissue. In contrast, the MSgt-FSgt promoter has the capacity to drive the reporter gene expression at a much higher level in all root cell types (Figure 6e and 7d). In summary, the MSgt-FSgt promoter activity was 9.5761.36, 2.8160.32 and 6.2961.31 times stronger in the root cortical cell, xylem and phloem tissues, respectively, compared to the 35S promoter (Table 3). After normalizing the activity of the CaMV35S promoter across the tissues, we observed high level of the MSgt-FSgt promoter expression in xylem (X), external phloem (EP), and internal phloem (IP) of stem; xylem (X) and lower epidermis (LE) of the leaf midrib; guard cell, subsidiary cells (SC) and palisade mesophyll (PM) of the leaf blade and xylem (X), phloem (P) and cortical cell (CC) of root (Figure 7e).

Comparison of promoter activity of MSgt-FSgt, FS3 and MS8 in vascular tissue
The GUS gene expression patterns in different vascular tissues and cell types of leaves, stems and roots of transgenic tobacco plants carrying the FS3, MS8 and MSgt-FSgt promoters were studied by CLSM. We observed that the MSgt-FSgt promoter was stronger than either of the parent promoters FS3 and MS8  (Table 4) higher activity than the FS3 and MS8 promoters in xylem tissue of the leaf midrib. Expression of MSgt-FSgt was found to be 4.6060.12, 2.1660.78, and 4.7261.11 times stronger than the FS3 promoter in external phloem, xylem and internal phloem tissues of the plant stem, respectively ( Table 4). The MSgt-FSgt promoter was also found to be 4.1960.42, 2.5660.32, and 3.88 60.32 times stronger than the MS8 promoter in external phloem, xylem and internal phloem tissues (Table 4). Similarly, the MSgt-FSgt promoter was 1.7660.63 and 2.3760.87 times stronger than the MS8 promoter in xylem and phloem tissues of the root, respectively. MSgt-FSgt also showed 1.6860.42 and 2.4560.61 fold stronger activity than the FS3 promoter in root xylem and phloem tissue (Table 4).

Nuclear protein binding assay of the MSgt-FSgt hybrid promoter
The interaction between tobacco nuclear proteins and the MSgt-FSgt promoter DNA was carried out as described in Methods. As shown in Figure 8a, specific DNA-protein binding (marked by an arrow) was observed in the presence of 5 mg and 10 mg of nuclear protein extract. The binding was completely abolished by competition with a 50 fold molar excess of unlabeled MSgt-FSgt DNA indicating that the interaction is specific (Figure 8a). This specific binding was not eliminated by competition with a 100 fold molar excess of FS3 but it was completely abolished by a 100 fold molar excess of the MS8 fragment (Figure 8b).
The promoter sequence where nuclear protein binds was identified by DNaseI foot printing analysis. It was a 33 bp long sequence that stretched between positions 2222 to 2189 (Figure 8c

Analysis of in vitro methylation assay of MSgt-FSgt and CaMV35S promoters
In silico analysis of MSgt-FSgt and CaMV35S promoter sequences using software (Support Vector Machine) identified a total number of 5 and 14 CG islands in MSgt-FSgt and CaMV35S promoter respectively (Figure 9a). The transient activities of methylated MSgt-FSgt and CaMV35S promoter in the tobacco protoplast system (Nicotiana tabaccum Xanthi brad) were determined as described in Methods. It was observed that methylated-MSgt-FSgt and CaMV35S promoter showed 64% and 79% less GUS activity compared to unmethylated MSgt-FSgt and CaMV35S promoter respectively (Fig. 9b). Statistical analysis revealed that the decrease in activity in the case of the methylated-MSgt-FSgt promoter (64%) was significant over decrease in the activity of the methylated-CaMV35S promoter (79%). The P value obtained was 0.0073 indicating a high level of significance.

Discussion
Several recombinant promoters were constructed by ligating different up-stream activation sequences of the MS8 promoter to the TATA-containing promoter sequence of the FS3 promoter. One recombinant promoter, MSgt-FSgt, developed in this study showed enhanced activity compared to wild-type promoters and the CaMV35S promoter [44][45][46][47][48] in tobacco protoplasts and transgenic tobacco and Arabidopsis plants. The observed increase in the relative activities of recombinant promoters may be due to the cooperative combinatorial interactions between the cis-elements present in the up-stream activation sequence (2306 to 2125) of MS8 and the TATA element of the FS3 promoter (FS-1). DNaseI foot-printing analysis revealed that a 33 bp long (between the coordinates 2222 to 2189) nuclear protein binding site (59AATAAGATGGCAAATGTGG TTTTCACATTACAC39) may be important for MSgt-FSgt promoter function, because deletion of this portion causes severe loss of MSgt-FSgt promoter activity as evidenced from the differences in activity between the MS-UAS3-FS1 promoter (construct No. 3) and the MS-UAS5-FS1 promoter (construct No. 5) (Figure 1).
In the present study, the MSgt-FSgt promoter showed 10.31 and 14.22 times stronger activity than CaMV35S promoter in the transient protoplast and transgenic tobacco systems respectively. In earlier reports as well as in the present study, it was demonstrated that the parental promoters MS8 [22] and FS3 [23] were 2 to 4 folds stronger than CaMV35S. However, Mirabilis mosaic virus full-length transcript promoter (MMV-FLt12) was reported to be 14 times stronger than CaMV35S in tobacco protoplast transient system [29] and about 25 times stronger than the CaMV35S in transgenic tobacco [26]. Based on these observations we may conclude that the MMVFLt12 promoter [26] is stronger than the MSgt-FSgt promoter. However, the recombinant promoter MSgt-FSgt is important because (i) it contains heterogeneous sequences compared to CaMV35S and MMVFLt12 (ii) it directed higher levels of constitutive gene expression in different plant cell types compared to the starting promoters (MS8 and FS3) and CaMV35S (iii) in a separate study by Kumar et al. (unpublished data), it was observed that the MSgt-FSgt promoter showed higher transgene activity under abiotic stress conditions (salt, salicylic/abscisic acid) compared to CaMV35S promoter.
Detailed analysis of GUS-specific transcripts isolated from transgenic plants containing CaMV35S, MS8, FS3 and MSgt-FSgt promoter constructs using Northern blot analysis, real-time PCR and qRT-PCR showed a fair correlation between the steadystate level of mRNA (transcript level) and GUS activity (protein level). Based on the above experimental evidence, it was confirmed that the activity of the MSgt-FSgt promoter was considerably stronger than that obtained from the CaMV35S, MS8 and FS3 promoters. Also it was noted that the MSgt-FSgt promoter directed reporter gene expression more consistently compared to the CaMV35S promoter in two different independent plant systems studied.
The intensities of blue fluorescence obtained from 4-MU (l ex 363 nm and l em 460 nm), the hydrolyzed product of MUG in the presence of the GUS enzyme, could be considered to be an indirect measure of relative promoter strength. 4-MU-intensity obtained from a particular cell/tissue type of a transgenic plant harboring the GUS-reporter gene (treated with 1 mM MUG solution) may reflect the cell/tissue specificity of a given promoter. The cell-specific expression of MSgt-FSgt was measured precisely using the analytical software 'LAS-AF' supplied with the confocal microscopy system (Leica, Germany), and was compared to that obtained from the MS8, FS3 and CaMV35S promoters. The normalized activity of MSgt-FSgt promoter clearly indicated that it is a near constitutive promoter and stronger than CaMV35S. The value obtained for the control sample (background) may be due to the presence of several candidates for blue fluorescence emissions; those are phenolic substances such as chlorogenic acid, caffeic acid, coumarines, ferulic acid, sinapic acid etc. All of these compounds emit blue fluorescence when excited at 405 nm [49].
The elevated vascular tissue-specific expression of the MSgt-FSgt promoter compared to the two wild-type promoters (FS3 and MS8) and the CaMV35S may be due to the presence of increased numbers of major plant cis-elements like Dof-1 and ARR1 in the MSgt-FSgt promoter in comparison to the wild-type promoters and CaMV35S. Dof-1 plays various roles in plant growth and development [50,51]. Recent studies showed involvement of Dof-1 in the vascular development of higher plants [52]. ARR1 and ARR2 factors usually act as transcriptional activators that promote the expression of a gene in plant cells through their own target sequences [53]. Besides the Dof-1 and ARR1 elements, the specific distribution pattern of the other cis-elements like Asf-1, G-Box etc. with different intervening spacer sequences in MSgt-FSgt promoter may also contribute to the vascular tissue specific and other functional role of the MSgt-FSgt promoter. Moreover, analyzing the MS8, FS3 and MSgt-FSgt promoter sequence by PlantPAN database [25] other cis-elements like AINTEGU-MENTA (ANT), ATHB-9 were also found to be increased in MSgt-FSgt promoter when compared to the two wild-type promoters (MS8 and FS3). Among these, ANT acts as a transcriptional activator playing a critical role not only in regulating ovule and female gametophyte development but also for gene expression in vegetative tissues [54]. The highest activity of MSgt-FSgt hybrid promoter in comparison to FS3 or MS8 in the transgenic system might be due to the increased presence of these cis-regulatory elements.
DNA methylation plays a crucial role in gene silencing [55]. Several studies have provided evidence that the level of gene expression from in vitro methylated DNA is strongly reduced when assayed in animal cells [56][57][58][59] and in plant cells [60][61][62][63], and this phenomenon could result in transgene silencing. We observed 64% decrease for MSgt-FSgt promoter is significant over 79% decrease for the CaMV35S promoter (P = 0.0073). In stably transformed independent transgenic Arabidopsis lines (T 1 genera-tion; 21 days old), it was also observed that the MSgt-FSgt and CaMV35S promoters resulted 6.7% and 13.3% of transgenic plants with minimum (basal) level of GUS activity respectively. This implies that both promoters are prone to be silenced but the degree of silencing could be higher in the case of the CaMV35S promoter compared to the MSgt-FSgt promoter.
There may be few possible constrains associated with using this promoter in plant genetic engineering. First, this recombinant promoter, being constitutive in nature, lacks tissue specificity. Second, in some situations, we may also need to restrict the expression level of a transgene, otherwise, it may cause problems to the plant itself, being a strong promoter, MSgt-FSgt, may not be ideal for use in such situations.
The observations of the present study revealed a promising future of MSgt-FSgt promoter as a potential candidate promoter for ectopic gene expression in plants. This study also highlighted the usefulness of CLSM in studying quantitative and qualitative gene expression in individual plant cells.