• Open Access

Functional analysis of cauliflower mosaic virus 35S promoter: re-evaluation of the role of subdomains B5, B4 and B2 in promoter activity

Authors


* Correspondence (fax 91-11-24115270; e-mail pburma@hotmail.com)

Summary

The cauliflower mosaic virus 35S (35S) promoter is used extensively for transgene expression in plants. The promoter has been delineated into different subdomains based on deletion analysis and gain-of-function studies. However, cis-elements important for promoter activity have been identified only in the domains B1 (as-2 element), A1 (as-1 element) and minimal promoter (TATA box). No cis-elements have been described in subdomains B2–B5, although these are reported to be important for the overall activity of the 35S promoter. We have re-evaluated the contribution of three of these subdomains, namely B5, B4 and B2, to 35S promoter activity by developing several modified promoters. The analysis of β-glucuronidase gene expression driven by the modified promoters in different tissues of primary transgenic tobacco lines, as well as in seedlings of the T1 generation, revealed new facets about the functional organization of the 35S promoter. This study suggests that: (i) the 35S promoter truncated up to –301 functions in a similar manner to the –343 (full-length) 35S promoter; (ii) the Dof core and I-box core observed in the subdomain B4 are important for 35S promoter activity; and (iii) the subdomain B2 is essential for maintaining an appropriate distance between the proximal and distal regions of the 35S promoter. These observations will aid in the development of functional synthetic 35S promoters with decreased sequence homology. Such promoters can be used to drive multiple transgenes without evoking promoter homology-based gene silencing when attempting gene stacking.

Introduction

The cauliflower mosaic virus 35S (35S) promoter has been extensively used to drive transgene expression in plants. This promoter was the focus of extensive studies attempting to delineate its functional domains and cis-elements therein in the late 1980s. Although first described by Franck et al. (1980), several groups (reviewed by Lam, 1994) have worked on the functional analysis of the promoter. Several domains of the 35S promoter were initially identified by deletion analysis (Odell et al., 1985, 1988; Ow et al., 1987; Benfey et al., 1989; Fang et al., 1989). The roles of the different domains were further analysed by gain-of-function studies, wherein individual domains were tested for their activities in transgenic plants (Benfey et al., 1990a,b). On the basis of these studies, the promoter was found to have a modular organization (Figure 1a) consisting of two main domains: A and B. Domain A was further subdivided into two subdomains: A1 and the minimal promoter (mp). Domain B, on the other hand, was subdivided into five subdomains: B1–B5. Benfey et al. (1989) observed that the individual subdomains of domain B could drive expression in distinct cell- and tissue-specific patterns only in conjunction with domain A (as summarized in table 1 and figure 4 of Benfey et al., 1990a). Domain A by itself exhibited high expression mainly in roots. On the basis of these observations, it was proposed that the near-ubiquitous expression of the 35S promoter was the result of a combinatorial effect of the various subdomains (Benfey and Chua, 1990; Benfey et al., 1990a,b). Later studies involved in the identification of the cis-elements present in these subdomains revealed the occurrence of binding sites of the transcription factors activating sequence factor-1 (ASF-1) (as-1 motif; Lam et al., 1989) and ASF-2 (as-2 motif; Lam and Chua, 1989) in subdomains A1 and B1, respectively, in addition to the TATA box in mp. Although the presence of other cis-elements, namely the CA-rich region in subdomain B3 (personal communication by T. Conner in Benfey and Chua, 1990; Lam, 1994), and the GTI and Dof factor binding sites in subdomain B4, have been hypothesized from time to time (Lam, 1994), their role in promoter activity has never been proven.

Figure 1.

(a) Modular organization of the cauliflower mosaic virus 35S (35S) promoter, as described by Benfey and Chua (1990). The 35S promoter consists of two domains: A and B. Domain A is further divided into the minimal promoter (mp) and subdomain A1. Domain B has five subdomains (B1–B5). (b) The ΔB5 promoter is a truncated (up to –301) version of the 35S promoter, wherein subdomain B5 has been deleted. (c) The bases that have been changed in the various (LS1–LS6) mutagenized 35S promoters by linker scanning are shown. The cis-elements found to be involved in 35S promoter activity are underlined. (d) Sequence of the subdomain B4 of 35S and of ΔB5IB4 showing the I-box-containing region used to replace subdomain B4 of the ΔB5 promoter. The I-box core in the 35S and ΔB5IB4 promoters is shown in bold. (e) Sequence of subdomain B2 in the promoter ΔB5ModB2.

More recently, promoters functionally analogous to 35S have been isolated and characterized from other caulimoviruses, e.g. mirabilis mosaic virus (MMV; Dey and Maiti, 1999) and figwort mosaic virus (FMV; Sanger et al., 1990; Maiti et al., 1997). It has been observed that these promoters show high sequence homology in the proximal region, but diverge in the upstream regions. Furthermore, the only common cis-elements present in similar positions in these three promoters are the as-1 and as-2 elements. An analysis of the observations made in earlier reports (Benfey and Chua, 1990; Benfey et al., 1990a,b) on the characterization of the 35S promoter led us to re-evaluate the importance of the subdomains B5, B4 and B2 in 35S promoter activity. We tested the effect of the deletion of subdomain B5 on 35S promoter activity. Furthermore, linker scanning (LS) mutagenesis was carried out for subdomain B4. Modified promoters were also developed wherein subdomain B4 was replaced with the I-box containing the region of the rbcS promoter, and subdomain B2 was replaced with a random DNA sequence. We thus developed several modified 35S promoters (summarized in Table 1 and represented in Figure 1b–e) and tested their functionality in transgenic tobacco lines. On the basis of the observations made in this study, in conjunction with earlier reports on the characterization of the 35S promoter, we propose the following: (i) subdomain B5 does not seem to be essential for overall promoter activity; (ii) the I-box and Dof factor binding sites in subdomain B4 seem to be important for 35S promoter activity; and (iii) subdomain B2 does not have any cis-elements, but may be essential for the appropriate placement of the flanking subdomains. Our work highlights new structural features of the 35S promoter.

Table 1.  Summary of modified promoters developed for analysis
PromoterSubdomain modifiedModification performed
ΔB5B5Subdomain B5 deleted from the cauliflower mosaic virus 35S (35S) promoter
ΔB5B4B5 and B4Subdomains B5 and B4 deleted from the 35S promoter, used as a control
LS mutantsB4Linker scanning (LS) mutations performed in subdomain B4 of the 35S promoter
LS1  
LS2  
LS3  
LS4  
LS5  
LS6  
ΔB5IB4B4Replaced subdomain B4 of ΔB5 with I-box containing region from rbcs1A promoter from Arabidopsis thaliana
ΔB5ModB2B5 and B2Replaced subdomain B2 of ΔB5 with a synthetic stretch

Results

Experimental design

In order to test the functionality of the different subdomains of domain B (B5, B4 and B2), 10 modified 35S promoters were developed (Table 1; Figure 1). To test the strength of these promoters, they were cloned upstream of the β-glucuronidase (gus) reporter gene (Figure 2). The GUS expression cassette was cloned into a binary vector pPZP200 (Hajdukiewicz et al., 1994) containing a nopaline synthase promoter (Pnos)-driven neomycin phosphotransferase II (nptII) gene. The nos-nptII expression cassette was used as a selection marker for plant transformation, as well as a reference gene for the normalization of plant-to-plant variations. The strength of the modified promoters was tested and compared with the 35S promoter in different tissues of primary tobacco transformants (T0), as well as in seedlings of the T1 generation. In order to reduce the influence of the physiological status of the plants on promoter activity, transgenic lines with promoters that were to be compared were grown and analysed simultaneously. While the transgenic lines with the 35S, ΔB5 and ΔB5IB4 promoter constructs were grown and assayed together, lines ΔB5ModB2 were grown together with ΔB5 and 35S as a separate set. The transgenic lines containing the LS promoter constructs were grown and analysed together with transgenic lines having 35S and ΔB5B4 promoter constructs as controls. The number of independent transgenic lines grown and analysed together as an experimental set is summarized in Table 2.

Figure 2.

Schematic representation of the binary vector developed for the transformation of tobacco. LB and RB are the left and right borders, respectively, of T-DNA in the binary vector. The selection marker gene (neomycin phosphotransferase II, nptII) is driven by the nopaline synthase (nos) promoter (Pnos) and contains the polyA region of the ocs gene (ocspA). The nptII gene was used as a selectable marker as well as a reference gene in the transgenic lines. Ptest denotes the promoter, whose expression is being analysed, driving the β-glucuronidase (gus) reporter gene with the polyA region of cauliflower mosaic virus 35S (35S polyA).

Table 2.  Number of transgenic lines with modified promoters grown and analysed as an experimental set
Experimental setTransgenic lines containing promotersNumber of transgenic lines tested
I35S24
ΔB525
ΔB5IB419
II35S19
ΔB5B431
LS122
LS224
LS319
LS423
LS525
LS619
III35S 6
ΔB5ModB2 9

Expression analysis of 35S promoter with deletion of subdomain B5 (ΔB5)

The subdomain B5 (–343 to –301) of the 35S promoter was deleted to create the promoter ΔB5 (Figure 1b). Twenty-five independent tobacco transgenic lines containing the ΔB5-gus construct and 24 lines containing the 35S-gus construct were developed and grown under the same conditions to test promoter activity in different tissues in transgenic plants. The promoter activity in leaves, as shown by the GUS activity, is represented in Figure 3a,b. The range of variation in GUS activity observed in independent transgenic lines containing the ΔB5-gus construct was similar to that recorded in lines containing the 35S-gus construct. This was also reflected in the range of normalized GUS activity (GUS activity/NPTII levels) observed with these two constructs, as summarized in a box-and-whisker plot (Figure 4a). The data were found to be normally distributed using a Kolmogorov–Smirnov Z-test, with mean values of 223.4 ± 23.0 and 299.8 ± 51.6 units for transgenic lines containing 35S-gus and ΔB5-gus constructs, respectively. A comparison of these mean values by t-test gave a two-sided P value of 0.12, indicating that the two means were not significantly different. The normalized GUS activity of the ΔB5 promoter in different tissues, namely stem, root, flower and callus, was also found to be similar to that of the 35S promoter (Figure 4b–f).

Figure 3.

β-Glucuronidase (GUS) activity of cauliflower mosaic virus 35S (35S) (a), ΔB5 (b) and ΔB5IB4 (c) promoters in the leaves of transgenic tobacco (T0) plants. The independent transgenic lines with the three different promoters were grown simultaneously under controlled conditions in a growth chamber (16 h day/8 h night; 28 ± 2 °C; relative humidity, 70%). Promoter activity was measured at two time points, i.e. from leaves of 45- and 60-day-old (after transfer to the soil) plants. Each bar represents the promoter activity of a single transgenic plant. Values are the means ± standard error of two independent experiments (with internal replicates).

Figure 4.

Box-and-whisker plots for comparison of normalized β-glucuronidase (GUS) activity [GUS activity/neomycin phosphotransferase II (NPTII) levels] in leaf (a), stem (b), root (c), flower (d) and callus derived from leaf (e) and from stem (f) of independent transgenic lines containing cauliflower mosaic virus 35S (35S), ΔB5 and ΔB5IB4 constructs. In the case of flower (d), only GUS activity was compared, as NPTII levels were undetectable. The horizontal lines in the box-and-whisker plots represent the 10th, 25th, 50th, 75th and 90th percentiles. The dotted line represents the mean of the data. Extreme values are depicted as small circles at the top and bottom of the plots.

To test whether the observations made in the primary transformants were also reflected in the next generation, GUS expression in the seedlings of representative transgenic lines containing the 35S-gus and ΔB5-gus constructs was quantified. Extracts made from approximately 150 7-day-old seedlings from each line were used to analyse promoter activity. The normalized GUS activities, represented in Figure 5, show that the activity of the ΔB5 promoter was similar to that of the 35S promoter.

Figure 5.

(a) β-Glucuronidase (GUS) activity of the cauliflower mosaic virus 35S (35S), ΔB5 and ΔB5IB4 promoters in the seedlings of the T1 generation obtained from independent primary transformants. Each bar represents the promoter activity observed in the extracts prepared from approximately 150 7-day-old seedlings from each line. (b) Comparison of the normalized GUS activity of the 35S, ΔB5 and ΔB5IB4 promoters in the seedlings of the T1 generation obtained from independent primary transformants.

Expression analysis of the 35S promoter with LS mutations (LS1–LS6) in subdomain B4

Six mutant 35S promoters, each encompassing an approximately 15-bp non-overlapping stretch of subdomain B4 of the 35S promoter, were developed using LS mutagenesis in order to delineate the functional cis-element(s) in B4 (Figure 1c). Several independent tobacco transgenic lines were developed with each LS-gus construct and grown simultaneously with 19 transgenic lines containing 35S-gus and 31 lines containing ΔB5B4-gus as controls (Table 2).

The GUS expression profiles in independent transgenic lines are presented in Figure 6. The mean GUS activity of ΔB5B4-gus-containing transgenic lines (163.2 ± 25.5 pmol MU/min/µg protein) was found to be significantly lower (t-test, P = 0.04) than that observed in transgenic lines containing the 35S-gus construct (265.0 ± 41.7 pmol MU/min/µg protein). In order to obtain a clearer picture of the comparison of activity between the transgenic lines with different constructs, we categorized the transgenic lines into those showing high and low expression levels of the gus gene (Table 3). Analysis of the data revealed that, although 32% of the lines containing the 35S-gus construct showed high expression, only 13% of the ΔB5B4-gus lines were high expressors. The transgenic plants containing LS1-gus, LS2-gus and LS4-gus constructs showed a similar distribution to plants containing 35S. However, lines containing LS3-gus and LS6-gus constructs behaved in a similar manner to the ΔB5B4 transgenic lines. The mean GUS activities of transgenic lines containing LS3 and LS6 constructs were found to be 240.6 ± 48.9 and 213.2 ± 40.3 pmol MU/min/µg protein, respectively. A t-test indicated that the means were not significantly different from those of ΔB5B4 (P = 0.17 and P = 0.30 for LS3 and LS6, respectively). Interestingly, lines containing the LS5-gus construct showed stronger promoter activity (mean GUS activity of 583.0 ± 111.8 pmol MU/min/µg protein) than those containing the 35S-gus construct (mean GUS activity of 261.0 ± 41.7 pmol MU/min/µg protein) (Figure 6g). This enhancement was also reflected in the observation that 60% of these plants expressed high levels of GUS activity (Table 3).

Figure 6.

β-Glucuronidase (GUS) activity of the cauliflower mosaic virus 35S (35S) (a), ΔB5B4 (b), LS1 (c), LS2 (d), LS3 (e), LS4 (f), LS5 (g) and LS6 (h) promoters in the leaves of transgenic tobacco (T0) plants. The independent transgenic lines containing the different promoters were grown simultaneously under controlled conditions in a growth chamber (16 h day/8 h night; 28 ± 2 °C; relative humidity, 70%). Promoter activity was measured from leaves of 45-day-old (after transfer to the soil) plants. Each bar represents the promoter activity of a single transgenic plant with internal replicates.

Table 3.  Expression analysis of transgenic lines containing cauliflower mosaic virus 35S (35S), ΔB5B4 and linker scanning (LS) promoter constructs. The transgenic lines are broadly grouped into high expressers [with β-glucuronidase (GUS) activity greater than 400 pmol methylumbelliferone (MU)/min/µg protein] and low expressers (with GUS activity lower than 400 pmol MU/min/µg protein), and represented as a percentage of the total number of independent transgenic lines analysed (shown in parentheses) for each construct
Transgenic linePercentage of plants showing high expressionPercentage of plants showing low expression
35S (19)3268
ΔB5B4 (31)1387
LS1 (22)3367
LS2 (24)3763
LS3 (19)1585
LS4 (23)3961
LS5 (25)6040
LS6 (19)1090

Expression analysis of ΔB5 promoter with a modified subdomain B4 (ΔB5IB4)

The ΔB5 promoter was further modified by replacing its subdomain B4 (–301 to –208) with a 73-bp region (–267 to –195) from the rbcS-1A gene promoter from Arabidopsis (Donald and Cashmore, 1990) to generate the promoter ΔB5IB4 (Figure 1d). The region used for replacement has two I-boxes (GATAAG). Nineteen independent transgenic lines were developed containing the ΔB5IB4-gus construct, and the strength of the promoter was compared with that of the 35S and ΔB5 promoters. The GUS activity observed in the leaves of primary transformants is presented in Figure 3c. A comparison of the normalized GUS activity of the ΔB5IB4 promoter with that of ΔB5 and 35S in various tissues of primary transformants is presented (box-and-whisker plots) in Figure 4. Our observations indicated that the overall range and distribution of the ΔB5IB4 promoter activity were similar to those of the 35S and ΔB5 promoters. This was also indicated by t-test analysis, wherein the mean GUS activity of transgenic lines containing the 35S promoter (765.6 ± 58.7 pmol MU/min/µg protein) was not significantly different (P = 0.11) from that of transgenic lines containing the ΔB5IB4 promoter. Thus, the I-box-containing region of the rbcS-1A gene promoter can functionally replace subdomain B4 in the ΔB5 promoter.

The activity of the ΔB5IB4-gus construct was also tested in seedlings of the T1 generation. Seedlings of 10 independent T0 lines were tested. These lines were representative of all the expression categories observed in the primary transformants. The GUS activity profile of the ΔB5IB4-gus-containing lines was similar to that of the 35S-gus- and ΔB5-gus-containing lines (Figure 5a). The normalized activity, however, showed a shift in the expression range. Although transgenic lines containing 35S showed a GUS activity in the range 31–190 units, most of the ΔB5IB4-containing seedlings had a normalized GUS level of less than 60 units (Figure 5b). The present observations therefore suggest that the I-box-containing region of the rbcS-1A gene promoter can functionally replace subdomain B4 in the mature parts of the plant, but not to the same extent in the seedlings (Figure 5).

Expression analysis of the 35S promoter with modified subdomain B2 (ΔB5ModB2)

In order to test the importance of subdomain B2 (–135 to –108) in 35S promoter activity, subdomain B2 of ΔB5 was replaced by a random sequence of 48 bp to create the ΔB5ModB2 promoter (Figure 1e). Eight independent transgenic lines were developed containing the ΔB5ModB2-gus construct and grown together with transgenic lines containing the 35S-gus and ΔB5-gus constructs (six transgenic lines each).

It was observed that the ΔB5ModB2 promoter showed a similar expression pattern to that of 35S in different tissues (leaf, stem, root and callus) of the primary transformants (Figure 7). Based on t-test analysis, the mean GUS activities observed in transgenic lines containing the 35S-gus construct were not found to be significantly different (P > 0.5) from those of lines containing the ΔB5ModB2-gus construct in the different tissues tested. The GUS activity was also comparable with that of the ΔB5 promoter (data not shown). The ΔB5ModB2-gus construct was observed to be as active as 35S-gus in the seedlings of the T1 generation (Figure 8). These observations suggest that subdomain B2 of the 35S promoter may not carry a cis-element of importance.

Figure 7.

(a) β-Glucuronidase (GUS) activity of the cauliflower mosaic virus 35S (35S) and ΔB5ModB2 promoters in the leaves of primary transgenic tobacco plants. The experimental design was the same as that described in Figure 3. Each bar represents the promoter activity of a single transgenic plant. Values represent the mean ± standard error of two independent experiments. Comparison of normalized promoter activity [GUS activity/neomycin phosphotransferase II (NPTII) levels] of 35S (WT) and ΔB5ModB2 in the leaf (b), stem (c), root (d) and callus derived from the leaf (e) and from the stem (f) of independent transgenic lines containing 35S and ΔB5ModB2 constructs represented as box-and-whisker plots.

Figure 8.

Comparison of β-glucuronidase (GUS) activity (a) and normalized GUS activity (b) of the 35S and ΔB5ModB2 promoter constructs in the seedlings of the T1 generation obtained from independent primary transformants. Each bar represents the promoter activity observed in the extracts prepared from approximately 150 7-day-old seedlings from each line.

Discussion

The 35S promoter was extensively analysed in the 1980s. The different subdomains of the 35S promoter were demarcated by deletion analysis or gain-of-function assays (Odell et al., 1985; Ow et al., 1987; Benfey et al., 1989; Fang et al., 1989). In the study by Fang et al. (1989), a deletion of the region upstream to –208 led to a 50% decrease in the transcriptional activity of the 35S promoter, thereby highlighting the significance of this region in promoter activity. In a later study by Benfey et al. (1990a), this region was subdivided into two regions, B5 and B4, spanning –343 to –301 and –301 to –208, respectively. There are no reasons given in the paper by Benfey et al. (1990a) for this division. No deletion analysis was carried out to examine the importance of B5 in the functioning of the 35S promoter. In a later gain-of-function study (Benfey et al., 1990b), a tetramer of subdomain B5 was fused upstream to domain A of the 35S promoter, and its activity was analysed in the different tissues of tobacco transgenic lines by histochemical localization of the GUS enzyme. Benfey et al. (1989) reported that ‘weak expression in mesophyll and vascular tissue is often observed in the leaf tissue’. Similarly, in the case of roots, they recorded some ‘apparent enhancement of expression’ in the cortex and vascular tissue. Weak expression was also reported in cells below the apical meristem in the stem, and no expression was observed in the petals of mature flowers (Benfey and Chua, 1990).

The present observations with the ΔB5 promoter suggest that subdomain B5 does not contribute significantly to the overall 35S promoter activity in the different tissues of the tobacco transgenic lines tested in this study. Although the present observations do not rule out the contribution of subdomain B5 in certain specific cell types, we believe that the expression pattern of subdomain B5, observed in the study by Benfey et al. (1990b), may possibly be a result of position effects. Indeed, in the conclusions to the above study (Benfey et al., 1990b), and in a subsequent report (Benfey and Chua, 1990), the activities of subdomains B4 and B5 have been referred to as a combination of B4 and B5 domain. However, it has never been reported that subdomain B5 may not be essential for the overall activity of the 35S promoter, and, to our knowledge, subdomain B5 is still considered to be an important functional domain.

Subdomain B4 extends from –301 to –208 of the 35S promoter. The 50% decrease in the promoter activity of 35S, observed by Fang et al. (1989) on deletion of the region upstream to –208, can only be attributed to subdomain B4, as our results show that the deletion of subdomain B5 has no effect on promoter activity. Although there has been no conclusive report of the presence of cis-elements in subdomain B4 that are important for the activity of the promoter, transcription factors, such as maize nuclear factor-1 (MNF-1), MNB1a and MNB1b from maize (Yanagisawa and Izui, 1992, 1993) and ASF-2 and GT-1 from tobacco (Lam, 1994), have been reported to bind to sequences in this domain, based on gel shift assays and in vitro competition experiments. Sequence analysis of this domain for the presence of cis-elements using the PLACE (plant cis-acting regulatory DNA elements) database (Higo et al., 1999) revealed the presence of several important cis-elements (Table S1, see ‘Supplementary material’).

In order to analyse the importance of these putative cis-elements, we carried out LS mutagenesis of subdomain B4. This revealed the importance of segments of the promoter spanned by linkers LS3 and LS6 (Figure 1c). LS3 (–256 to –270) contained a binding site for Dof factor, characterized by the presence of the core element ‘AAAG’, whereas LS6 (–209 to –226) contained an I-box element. The LS3 linker lies within the –281 to –235 region of the 35S promoter. In a study by Yanagisawa and Izui (1992), the MNF-1 transcription factor from maize was shown to bind to the –281 to –235-bp region of the 35S promoter. MNF-1 was later identified to be the Dof domain transcription factor, and is known as the Dof1 protein of maize. In another study (Zhang et al., 1995), the transcription factor OBF binding protein-1 (OBP-1) from Arabidopsis has been shown to bind to the AAGG motif in the –281 to –235-bp region of the 35S promoter. This early evidence for the presence of a Dof binding site in 35S was not confirmed further by mutational analysis of the 35S promoter. To our knowledge, this is the first study in which mutation of the Dof protein binding site of 35S has demonstrated its involvement in promoter activity.

The present study also revealed the role of the I-box core present in the –209 to –226 region (LS6) of the 35S promoter. The I-box elements are found in light-regulated promoters, such as rbcS, cab and nia, and have been shown to bind to I-box binding factors (IBFs) (Borello et al., 1993). In order to substantiate this observation, we replaced subdomain B4 with an I-box-containing domain from another functional promoter (rbcS-1A) from Arabidopsis to create the ΔB5IB4 promoter. This replacement strategy effectively leads to disruption of most of the putative cis-elements of subdomain B4, except the I-box. The ΔB5IB4 promoter was found to be functionally equivalent to the 35S promoter in all the tissues tested. We have shown previously that synthetic promoters can be created by ‘domain swapping’, wherein the domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters (Bhullar et al., 2003). Our results thus demonstrate that the domain from rbcS-1A containing a functional I-box in its native context can successfully replace the activity of subdomain B4.

The up-regulation of promoter activity observed in LS5 construct-containing transgenic lines may be a result of either removal of a negative regulatory element or an inadvertent incorporation of a CCAAT box that is frequently observed in plant promoters (Sawant et al., 2001).

Analysis of the observations made by Fang et al. (1989) revealed that subdomain B2 possibly accounts for ~20% of 35S promoter activity. This is based on their observation that deletion of the 35S promoter upstream to –168 reduces promoter activity to 30%, and, when the deletion is extended to –105, there is a drastic fall in promoter activity, with the deleted promoter showing only 10% of the wild-type promoter activity. Therefore, the overlapping deleted region (comprising subdomain B2) would account for 20% activity. However, in a ‘gain-of-function’ study by Benfey et al. (1990b), it was reported that, when subdomain B2 was combined with domain A, there was no change in expression pattern in comparison with a situation in which domain A was taken alone. Gel mobility shift assays on subdomain B2 did not show any binding of transcription factors to this region (Lam, 1994). These observations have led us to hypothesize that subdomain B2 has no transcription factor binding sites, although it may be essential for maintaining the appropriate distance between the proximal and distal components of the 35S promoter. We therefore tested the effect of replacing this subdomain with a 48-bp novel synthetic stretch of DNA. Our observations revealed that the ΔB5ModB2 promoter functioned in a similar manner to 35S. Thus, the decrease in promoter activity observed in the deletion experiments may have been a result of a disruption in organization, i.e. appropriate spacing of the proximal and distal parts of the 35S promoter, rather than the subdomain having a functional cis-element.

Sequence analysis of the subdomains investigated in this study revealed the presence of several putative cis-elements (Table S1) based on the plant cis-element database PLACE (Higo et al., 1999). The Dof core sequence is the most common element found in the analysed subdomains, although those present in subdomains B5 and B2 do not seem to be essential for promoter activity. The present study, based on an analysis of the 35S promoter, suggests that: (i) the 35S promoter truncated up to –301 functions in a similar manner to the –343 (full-length) 35S promoter; (ii) the Dof core and I-box core observed in subdomain B4 contribute to 35S promoter activity; and (iii) subdomain B2 is essential for maintaining an appropriate distance between the proximal and distal parts of the 35S promoter in the tissues and conditions tested in the present study. Our results therefore highlight new facets of the functional organization of the 35S promoter, a promoter used extensively in plant biotechnology.

The analysis of these subdomains of 35S is also significant in view of our work on the identification of strategies to develop synthetic promoters (Bhullar et al., 2003). These promoters can be used to drive different transgenes without the problem of promoter homology-based gene silencing when attempting gene stacking (Gurr and Rushton, 2005; Halpin, 2005). In a previous study (Bhullar et al., 2003), using domain A of the 35S promoter as a model, we demonstrated that functionally equivalent promoters with minimum sequence homology could be developed by modifying sequences present between cis-elements of a promoter or by domain swapping. The observations made in the present study will thus aid in the development of a functionally equivalent synthetic 35S promoter with minimum homology to its wild-type counterpart.

Experimental procedures

Assembly of modified promoters

Modified promoters (Table 1) were assembled by polymerase chain reaction (PCR), followed by subsequent cloning in pPCR Script SK(+) (Stratagene, La Jolla, CA, USA). The promoter ΔB5 was amplified from the 35S promoter using primers flanking subdomain B4 and the +1 site of the 35S promoter. The ΔB5IB4 promoter was synthesized using recursive PCR (Dillon and Rosen, 1990), whereas the ΔB5ModB2 and LS constructs were assembled using splicing by overlap extension (SOE-ing) reactions (Horton, 1993). The DNA sequences of the modified promoters were confirmed by sequencing. Standard cloning procedures were followed, as described in Sambrook et al. (1989). The modified promoters (Ptest) (Figure 2) were cloned as HindIII-NcoI fragments upstream to the gus reporter gene with a 35S polyA signal in pPCR Script SK(+). The Ptest gus expression cassette thus developed was cloned as a SacI-SalI fragment in the binary vector pPZP200 (Hajdukiewicz et al., 1994), containing a Pnos-driven nptII gene and a chloramphenicol acetyl transferase (cat) gene under the control of the 35S (P35S) promoter at EcoRI and HindIII sites, respectively (Figure 2), in all constructs except LS constructs used in Experimental set II (Table 2). These vectors did not carry the 35S-cat expression cassette.

Development of transgenic lines

Binary vectors were mobilized into the disarmed Agrobacterium strain GV2260 by electroporation. The transgenic lines were developed in Nicotiana tabacum cv. Xanthi. Agrobacterium-mediated transformation of leaf disc explants was carried out following the protocol of Svab et al. (1995). With each construct, 6–27 independent transgenic lines were developed. Transgenic lines were grown in a growth chamber (16 h day/8 h night; 28 ± 2 °C; relative humidity, 70%). Leaves were harvested from 45- and 60-day-old (after transfer to soil) plants, and flowers were harvested from transgenic plants when pollen dispersal was at its optimum.

For the analysis of expression in stem and root, the plants were grown in a tissue culture room (16 h day/8 h night; 28 ± 2 °C) in glass bottles, and stem and root were harvested 35–40 days following subculturing. Callus was raised from stem and leaf tissue by placing the explants on Murashige–Skoog (MS) agar supplemented with 2 mg/L naphthaleneacetic acid (NAA) and 0.5 mg/L benzylaminopurine (BAP). Extracts were prepared after 14–16 days of callusing.

Selfed (T1) seeds were collected from growth chamber-grown primary transformants (T0) and germinated on germination paper. Approximately 150 7-day-old seedlings were taken to prepare total protein extracts.

Enzyme assays for the estimation of promoter strength

Total protein from different tissues of transgenic plants or seedlings was extracted in GUS extraction buffer (Jefferson, 1987). The protein concentration was estimated following Bradford (1976). Fluorometric GUS assays using 4-methylumbelliferyl-β-d-glucuronide (MUG) substrate were performed according to Jefferson (1987). The product released (MU) was estimated using a DyNA Quant 200 Fluorometer (Hoefer Pharmacia Biotech, San Francisco, CA, USA). GUS activity was expressed as picomoles of MU per minute per microgram of protein.

The amount of NPTII protein in total protein extracts was measured by enzyme-linked immunosorbent assay (ELISA) using a kit obtained from Agdia Incorporated, Elkhart, IN, USA (Cat. No. PSP 73000).

Statistical analysis of the data

The data on the GUS activity obtained in transgenic lines with different constructs were observed to be normally distributed by the Kolmogorov–Smirnov Z-test performed using the Statistical Package for Social Sciences Version 11.0. A t-test was carried out to determine whether the observed mean values of GUS activity in transgenic lines with different promoter constructs were similar or significantly different.

Acknowledgements

This work was supported by the Department of Biotechnology, Government of India and grants from Department of Science and Technology – Funds for Improvement of Science and Technology Infrastructure (DST-FIST) and University Grants Commission – Special Assistance Programme (UGC-SAP) to the parent department. SB, SD and SC were supported by Research Fellowships from the Council of Scientific and Industrial Research, Government of India. SA was supported by a Bakshi Research Fellowship, University of Delhi South Campus (UDSC).

Ancillary