BACKGROUND
enhancers are dna elements that are able to increase transcription from other promoters whether they are placed upstream or downstream of transcription start sites and their promoter enhancing activity is independent of orientation relative to the transcription start site  <cit> .
enhancers that are effective in plants have been isolated from genes of plants as well as from genes of viruses and bacteria that infect plants.
these include enhancers from the tobacco tcup  <cit> , the pea plastocyanin  <cit> , the cauliflower mosaic virus 35s   <cit> , the figwort mosaic virus  <cit>  and the agrobacterium tumefacians  <dig>  <cit>  and ocs promoters  <cit> .
plant virus-derived promoters have been shown to be a rich source of strong constitutive promoters for use in plant biology and several have been shown to contain enhancer sequences  <cit> .
the camv 35s promoter has been used extensively in driving transgenes in transgenic plants.
many other viral promoters have also been shown to effectively drive expression of transgenes; camv 19s, rice tungro bacilform virus   <cit> , soybean chlorotic mottle virus  <cit> , mirabilis mosaic virus  <cit> , figwort mosaic virus   <cit> , peanut streak chlorotic virus  <cit> , banana streak badnavirus  <cit> , cestrum yellow leaf curling virus   <cit>  and sugarcane bacilliform badnavirus   <cit> .
among these, the camv 35s and the fmv promoters have been demonstrated to have enhancer sequences within the promoter  <cit> .
the camv 35s enhancer  <cit>  is the most common enhancer used in plant biology.
several studies have shown that the camv 35s promoter is not as active as other strong constitutive promoters in monocots  <cit> , raising the question whether the camv 35s enhancer sequences are as effective in monocots as they are in dicots.
however, 2x and 4x arrays of the camv 35s enhancer have been shown to enhance transcription of heterologous promoters in stable transformants of rice as well as to cause increased transcript accumulation of endogenous genes  <cit> .
the sugarcane bacilliform virus , like the cauliflower mosaic virus, is in the caulimoviridae family of viruses.
while the cauliflower mosaic virus is in the caulimovirus genus and mostly infects dicots, scbv is in the badnavirus genus along with commelina yellow mottle virus  and rtbv and infects monocots  <cit> .
badnaviruses have circular genomes that produce a terminally redundant transcript  <cit> .
like comv, scbv has three large open reading frames on its plus strand  <cit> .
promoters from several badnaviruses have been shown to drive expression of heterologous genes in transgenic plants  <cit> .
because these viruses infect monocots, they may be useful sources of strong promoters for monocots.
scbv promoters from several isolates of the virus have been tested in transgenic plants and shown to be highly expressed in most tissues tested  <cit> .
we present a characterization of an 839 bp fragment of the sugarcane bacilliform virus - ireng maleng isolate  promoter and demonstrate that it is comparable in strength to the strong maize ubiquitin  <dig>  promoter in transgenic maize.
this work also presents a dissection of the scbv-im promoter and the identification of sequences that can enhance transcription when placed upstream of a truncated maize alcohol dehydrogenase  promoter.
similar to what was seen with the camv 35s enhancer  <cit> , multiple tandem copies of the scbv-im enhancer are more effective in increasing transcription than a single copy.
an activation tagging element containing four tandem copies of the enhancer element has been introduced into maize.
examination of events containing the activation tagging element indicates that the 4x scbv-im enhancer is capable of causing an increase in accumulation of transcripts of native maize genes near the site of insertion of the scbv-im enhancer.
RESULTS
the scbv-im promoter is a strong promoter in transgenic maize
to compare the strength of the scbv-im promoter relative to a known strong promoter, transgenic plants containing scbv-im::aad <dig> and zmubi1::aad <dig> transgenes were generated.
aad <dig> encodes an enzyme that degrades  <dig> -d and aryloxyphenoxypropionate herbicides and plants expressing this gene are tolerant of these herbicides  <cit> .
transcript accumulation was measured in samples from various tissues of t <dig> plants by rt-qpcr.
tissue samples were taken from the youngest fully expanded leaf at the v <dig>  and v <dig> stages, from the leaf below the developing ear at the r <dig> stage, from a 1 cm section from the tip of a root at v <dig> and v <dig> stages and from the tassels at r <dig> stage.
figure  <dig> shows aad <dig> transcript accumulation in  <dig> events containing each transgene.
in the leaf samples collected at v <dig> stage, plants containing the scbv-im::aad <dig> transgene accumulate more transcript than plants containing zmubi1::aad <dig> , at the v <dig> stage, leaves accumulate similar amounts of aad <dig> transcripts  while in the leaf samples of r <dig> plants lower levels of aad <dig> transcript accumulate in the scbv-im::aad <dig> transgenic plants .
in the roots of v <dig> and v <dig> plants, scbv-im::aad <dig> transgenic plants accumulate more of the aad <dig> transcripts than zmubi1::aad <dig> transgenic plants .
in tassel tissues, zmubi1::aad <dig> transgenic plants accumulate more aad <dig> transcript.
these results demonstrate that the scbv-im promoter is stronger or comprable in strength to the strong, constitutively expressed maize ubiquitin  <dig> promoter  <cit>  in young leaf and root tissues, but is weaker in the leaf below the devloping ear and in tassel tissues at r <dig> figure  <dig>
comparison of the scbv-im and zmubi <dig> promoters in transgenic maize tissues.
transgenic, single copy maize events containing scbv-im::aad <dig> and zmubi1::aad <dig> constructs were analyzed for aad <dig> transcript accumulation in the youngest fully expanded leaf of v <dig>  and v <dig>  stage plants, the leaf just below the ear of r <dig> plants , a 1 cm section from the tip of the root in v <dig>  and v <dig>  stage plants, and in the tassels of r <dig> stage plants .
three events for each of the two constructs were compared by rt-qpcr using primers specific to the aad <dig> transcript and normalized to an endogenous transcript, tip  or maz <dig> .
the error bars represent the standard deviation of three measurements.
analysis of variance  indicate that more aad <dig> transcript accumulate in the scbv-im::aad <dig> events than in zmubi1::aad <dig> events in leaves of v <dig> stage plants, the roots of v <dig> and v <dig> stage plants, while similar levels of aad <dig> transcript accumulate in leaves of v <dig> stage plants.
zmubi1::aad <dig> transgenic plants accumulate more aad <dig> transcript than scbv-im::aad <dig> transgenic plants in leaf and tassel tissues at r <dig>
scbv-im enhancer identification and characterization
the activity of the scbv-im enhancer was demonstrated in transient assays first by identifying sequences that are necessary for high levels of transcription and then by identifying sequences that can enhance transcription from a heterologous promoter.
the sequence of the scbv-im promoter is shown in figure 2; the transcription start site was mapped by 5′ race.figure  <dig>
scbv promoter sequence  and fragments used in experiments .
the transcription start site was mapped by 5′ race and sequences in the 5′ untranslated region of the transcript are underlined .
the putative tata box is underlined at position − <dig> – - <dig>  the scbv-im enhancer sequences are in bold.
the fragments of the scbv-im promoter used in transient assays are displayed.
the position of the transcription start site  is shown.
scbv-im promoter fragments scbv <dig>  scbv <dig> and scbv <dig>  were cloned upstream of the luciferase  reporter gene.
transcriptional activities of these constructs were tested by transfecting maize hi-ii suspension cells and monitoring relative activities of the reporter genes.
to test the activities of different scbv-im promoter fragments, equal molar concentrations of the test plasmids and a reference plasmid , used as an internal control to normalize for differences in transformation efficiency, were co-introduced into maize hi-ii suspension cell cultures by particle bombardment.
two days after the bombardment, total protein was isolated from transfected cells and luc and gus enzymatic activities were determined.
activity was expressed as the ratio of luc to gus activity.
the results show that the promoter fragment scbv <dig> had 60% of the activity of the scbv <dig> promoter fragment  and the scbv <dig> promoter fragment had only 10% of the activity of the scbv <dig> fragment.
these data indicate that sequences necessary for most of the scbv-im promoter activity reside upstream of the 333 bp fragment.figure  <dig>
promoter analysis of the scbv-im promoter.
three fragments of the scbv-im promoter fused with the luciferase reporter gene and tested for activity in transient assays and compared with a luciferase construct without a promoter.
these constructs were transfected into maize hi-ii cultures along with a zmubi1::gus construct.
activity is reported as the ratio of luciferase activity  from the test construct to gus activity  from the internal control construct.
error bars represent the standard deviation of three measurements.
analysis of variance and tukey-kramer hsd tests  indicate significant differences in lu/gu ratios for all constructs tested.
next, two upstream fragments of the scbv-im promoter were tested for their ability to enhance transcription from a truncated heterologous promoter.
two fragments of the scbv-im promoter  were cloned upstream of a truncated maize alcohol dehydrogenase  <dig>  promoter   <cit>  fused to the firefly luciferase gene.
these constructs were designated scbv282::zmadh1::luc and scbv537::zmadh1::luc, respectively.
maize hi-ii suspension cells were transfected with plasmids scbv282::zmadh1::luc, scbv537::zmadh1::luc and zmadh1::luc  along with the reference plasmid containing zmubi1::gus.
overall, these promoters gave much lower activity than the intact scbv promoter .
this may be because the zmadh <dig> chimeric promoters are inherently weaker than the intact scbv-im promoter or because a promoter fragment of the scbv-im promoter necessary for high activity was not included in the chimeric constructs.figure  <dig>
enhancer analysis of the scbv-im promoter.
two fragments of the scbv-im promoter were fused with the truncated zmadh <dig> promoter and the luciferase reporter gene.
these constructs were tested for activity in transient assays and compared with a construct containing only the minimal zmadh <dig> promoter.
these constructs were transfected into maize hi-ii cultures with a zmubi1::gus construct.
activity is reported as the ratio of luciferase activity  from the test construct to gus activity  from the internal control construct.
error bars represent the standard deviation of three measurements.
analysis of variance and tukey-kramer hsd tests  indicate significant differences in lu/gu ratios for all constructs tested.
the results shown in figure  <dig> indicate that the scbv <dig> fragment  was able to enhance activity of the truncated zmadh <dig> promoter more effectively than the larger scbv <dig> fragment .
these results indicate that these fragments of the scbv-im promoter cause an increase in activity of the reporter gene driven by a truncated heterologous promoter, and that most of this enhancing activity lies within the −434 bp to −153 bp region.
to determine whether multiple copies of the scbv-im enhancer are more effective in activating transcription,  <dig>   <dig> and  <dig> copies of the scbv <dig> fragment were cloned upstream of the truncated zmadh <dig> promoter fused to the luc gene and bombarded into maize hi-ii suspension cells.
constructs containing  <dig>   <dig> and  <dig> tandem copies of the scbv-im enhancer had  <dig> times,  <dig> times and  <dig> times more activity, respectively, than did cells bombarded with the zmadh1::luc construct without any scbv-im sequences .
it should be noted that in the experiment shown in figure  <dig>  the activity of scbv282::zmadh1::luc and zmadh1::luc was substantially greater than in the experiment shown in figure  <dig>  however, similar variation in promoter activity between independent experiments conducted with different cell preparations has previously been reported  <cit> .figure  <dig>
analysis of scbv-im enhancer arrays.
tandem copies  of the scbv-im enhancer were fused with the truncated zmadh <dig> promoter and the luciferase reporter gene.
these constructs were tested for activity in transient assays and compared with a construct containing only the minimal zmadh <dig> promoter.
these constructs were transfected into maize hi-ii cultures with a zmubi1::gus construct.
activity is reported as the ratio of luciferase activity  from the test construct to gus activity  from the internal control construct.
error bars represent the standard deviation of three measurements.
analysis of variance and tukey-kramer hsd tests  indicate significant differences in lu/gu ratios for all constructs tested.
scbv-enhancer activity in stable maize transformants
to determine whether the scbv-im enhancer can increase expression of genes within the maize genome, an activation tagging construct consisting of the 4x tandem array of the scbv <dig> enhancer  and a selectable marker composed of the rice actin promoter driving expression of the aad <dig> herbicide resistance gene and the maize lipase 3′ utr  <cit>  was cloned and transformed into maize plants via agrobacterium-mediated transformation.figure  <dig>
schematic of enhancer construct, peps <dig>  used in maize transformation.
the construct includes an array of  <dig> copies of the scbv-im enhancers  and the aad <dig> herbicide resistance gene  containing the rice actin promoter fused with the aad <dig> gene and the maize lipase 3′ utr relative to the left border  and right border  of agrobacterium t-dna.
transformants were examined for the location of the t-dna insertion and the proximity of the enhancers to annotated genes in the maize genome that were reported to be expressed at moderate levels in leaf tissues  <cit> .
determination of the site of integration of the construct was attempted for  <dig> events by a transgene border sequence identification method  <cit>  and  <dig> of these events were mapped to locations in the maize b <dig> reference genome.
to determine whether the enhancers within the t-dna sequence are able to cause an increase in transcript accumulation of endogenous genes, these transformants were examined to identify t-dna insertion sites within ~ <dig>  kb of a gene.
the camv 35s enhancer has been demonstrated to up-regulate genes within ~ <dig> kb of the enhancer sequences  <cit> .
eleven events were examined to determine whether transcripts of genes adjacent to the activation tagging element were more abundant in the transgenic events than non-transgenic control plants.
for five of these genes , no transcripts were detected in either the transgenic or non-transgenic lines.
for the six other genes, transcripts were detected from genes adjacent to the activation tagging element.
in these events, two genes  showed a similar level of these transcripts in transgenic and non-transgenic plants .
however, in  <dig> other events the transcripts of genes adjacent to the activation tagging element were more abundant in the transgenic plant than the non-transgenic plant.
for two genes , the transgenic plant showed more transcript than did the non-transgenic plant; this increase in abundance was  <dig>  and  <dig>  fold, respectively.
for the other two genes , no transcript was detectable in the non-transgenic plant but transcript was clearly detectable in the transgenic plant.
these results demonstrate the 4x tandem array of the scbv-im enhancer can increase transcript abundance in a stable transformed maize plant and in some cases may cause ectopic expression of genes that are not expressed, or expressed at very low levels.
it also indicates that the 4x tandem array of the scbv-im enhancer meets the traditional definition of an enhancer  <cit>  because it can function upstream or downstream of the transcription unit and in either orientation.figure  <dig>
analysis of enhancer activity in transgenic maize plants.
the relative locations of the activation tagging element and the gene examined are shown.
the activation tagging element is represented by four yellow boxes representing the 4× scbv enhancer tandem array and the blue arrow representing the aad <dig> selectable marker cassette.
maize genes are represented by the green box with the transcription start site and direction represented by the arrow.
distance to promoter reflects the estimated distance from the enhancers to the translational start site of the gene.
the relative transcript abundance is the amount of transcript of the gene in transgenic plants compared with the level of the transcript in non-transgenic plants.
an asterix indicates that the values were significantly different as determined by a t-test .
discussion
the promoter sequences that we define include significant portions of the scbv orf iii gene.
the scbv <dig> sequence, which has the greatest activity in transient assays, overlaps with  <dig> protein coding nucleotides.
the sequences overlapping the orf iii gene contain most of the promoter activity as demonstrated by the scbv <dig> fragment containing only 20 bp of the orf iii gene and having just 10% of the promoter activity of the scbv <dig> sequence .
the scbv <dig> enhancer fragment contains 189 bp of orf iii coding sequence.
a similar situation is found in arabidopsis where regulatory elements for the promoter of zwichel  gene are found in exon and intron sequences of the adjacent hydroxyisobutyrl-coa hydrolase  <dig>  gene  <cit> .
the enhancer sequences in the scbv-im promoter were able to increase the activity of the truncated zmadh <dig> promoter , but these chimeric promoters were much weaker than the intact scbv-im promoter in the transient assays .
this difference is so great it is unlikely to be the result of different cell preparations and may be the result of the scbv-im upstream activating sequences interacting differently with the heterologous core zmadh <dig> promoter and the native scbv-im promoter.
similar results were seen when the camv 35s enhancer was placed upstream of the camv 19s core promoter  <cit> .
deletion analysis of the scbv-im promoter showed that removing sequences from − <dig> to − <dig> caused a 30% decline in promoter activity, while removing sequences from − <dig> to − <dig> caused a 90% decline in activity .
it was, therefore, somewhat surprising to observe that the chimeric promoter scbv537::zmadh <dig>  had less activity than the chimeric promoter scbv282::zmadh <dig>  since the longer fragment in the promoter deletion analysis had the most activity.
surprising results are often obtained when portions of promoters are added or deleted and even small portions of promoters can have dramatic effects.
for example, dey and matti  <cit>  showed that removing 50 bp of the mmv promoter increased activity  <dig> fold and simon et al.
<cit>  showed that deleting 54 bp of the inner no outer promoter of arabidopsis could reverse a silenced promoter.
multiple copies of the scbv-im enhancer cause an increase in activity of the chimeric promoters in transient assays .
this is consistent with what has been observed with the camv 35s and fmv ehancers  <cit>  and may be due to multiple copies of the enhancer being more efficent in recruiting transcription factors to the promoter.
the scbv <dig> fragment is capable of acting as a transcriptional enhancer when present in the maize genome in a 4x tandem array.
in events containing the 4x scbv-im enhancer upstream and downstream of genes, and in either orientation with respect to these genes, increased transcript accumulation was observed .
furthermore, the element appeared to cause accumulation of transcripts that are not present, or present in very low levels, in non-transgenic lines.
this demonstrates that the scbv-im enhancer may be used for activation tagging in maize.
the scbv-im enhancers increased expression of  <dig> out of  <dig> genes that showed non-detectable expression in non-transgenic control plants.
this is similar some studies that have reported ectopic expression of genes when the camv 35s enhancers integrate nearby  <cit> .
CONCLUSIONS
in this work, we demonstrate that the scbv-im promoter is comparable in strength to the zmubi <dig> promoter in transgenic young maize leaves and roots and we identify sequences from the scbv-im promoter that can function as a transcriptional enhancer in maize plants.
we used transient assays to identify promoter sequences that are responsible for most of the promoter activity and sequences of this promoter that enhance expression from a heterologous promoter.
finally, we generated stable transgenic plants containing 4x tandem arrays of the scbv-im enhancer and demonstrated that transcripts of genes near the insertion site are more abundant than in non-transgenic control plants.
activation tagging by randomly inserting transcriptional enhancers in the genome is a powerful tool for identifying gene function.
the camv 35s enhancer has been used to develop activation tagging systems for arabidopsis, rice and barley.
using these activation tagging systems, researchers have identified a number of genes with novel functions  <cit> .
to date, no activation tagging system has been developed for maize.
as a first step in developing an activation tagging system for maize, we have identified a transcriptional enhancer from scbv-im and have shown it to be able to activate transcription from a truncated zmadh <dig> promoter in transient assays and from endogenous promoters in transformed maize plants.

 enhancers are dna elements that are able to increase transcription from other promoters whether they are placed upstream or downstream of transcription start sites and their promoter enhancing activity is independent of orientation relative to the transcription start site. enhancers that are effective in plants have been isolated from genes of plants as well as from genes of viruses and bacteria that infect plants. these include enhancers from the tobacco tcup, the pea plastocyanin, the cauliflower mosaic virus 35s, the figwort mosaic virus and the agrobacterium tumefacians and ocs promoters. plant virus-derived promoters have been shown to be a rich source of strong constitutive promoters for use in plant biology and several have been shown to contain enhancer sequences. the camv 35s promoter has been used extensively in driving transgenes in transgenic plants. many other viral promoters have also been shown to effectively drive expression of transgenes; camv 19s, rice tungro bacilform virus, soybean chlorotic mottle virus, mirabilis mosaic virus, figwort mosaic virus, peanut streak chlorotic virus, banana streak badnavirus, cestrum yellow leaf curling virus and sugarcane bacilliform badnavirus. among these, the camv 35s and the fmv promoters have been demonstrated to have enhancer sequences within the promoter. the camv 35s enhancer is the most common enhancer used in plant biology. several studies have shown that the camv 35s promoter is not as active as other strong constitutive promoters in monocots, raising the question whether the camv 35s enhancer sequences are as effective in monocots as they are in dicots. however, 2x and 4x arrays of the camv 35s enhancer have been shown to enhance transcription of heterologous promoters in stable transformants of rice as well as to cause increased transcript accumulation of endogenous genes. the sugarcane bacilliform virus, like the cauliflower mosaic virus, is in the caulimoviridae family of viruses. while the cauliflower mosaic virus is in the caulimovirus genus and mostly infects dicots, scbv is in the badnavirus genus along with commelina yellow mottle virus and rtbv and infects monocots. badnaviruses have circular genomes that produce a terminally redundant transcript. like comv, scbv has three large open reading frames on its plus strand. promoters from several badnaviruses have been shown to drive expression of heterologous genes in transgenic plants. because these viruses infect monocots, they may be useful sources of strong promoters for monocots. scbv promoters from several isolates of the virus have been tested in transgenic plants and shown to be highly expressed in most tissues tested. we present a characterization of an 839 bp fragment of the sugarcane bacilliform virus - ireng maleng isolate promoter and demonstrate that it is comparable in strength to the strong maize ubiquitin promoter in transgenic maize. this work also presents a dissection of the scbv-im promoter and the identification of sequences that can enhance transcription when placed upstream of a truncated maize alcohol dehydrogenase promoter. similar to what was seen with the camv 35s enhancer, multiple tandem copies of the scbv-im enhancer are more effective in increasing transcription than a single copy. an activation tagging element containing four tandem copies of the enhancer element has been introduced into maize. examination of events containing the activation tagging element indicates that the 4x scbv-im enhancer is capable of causing an increase in accumulation of transcripts of native maize genes near the site of insertion of the scbv-im enhancer.  the scbv-im promoter is a strong promoter in transgenic maize to compare the strength of the scbv-im promoter relative to a known strong promoter, transgenic plants containing scbv-im::aad and zmubi1::aad transgenes were generated. aad encodes an enzyme that degrades -d and aryloxyphenoxypropionate herbicides and plants expressing this gene are tolerant of these herbicides. transcript accumulation was measured in samples from various tissues of t plants by rt-qpcr. tissue samples were taken from the youngest fully expanded leaf at the v and v stages, from the leaf below the developing ear at the r stage, from a 1 cm section from the tip of a root at v and v stages and from the tassels at r stage. figure  shows aad transcript accumulation in events containing each transgene. in the leaf samples collected at v stage, plants containing the scbv-im::aad transgene accumulate more transcript than plants containing zmubi1::aad, at the v stage, leaves accumulate similar amounts of aad transcripts while in the leaf samples of r plants lower levels of aad transcript accumulate in the scbv-im::aad transgenic plants. in the roots of v and v plants, scbv-im::aad transgenic plants accumulate more of the aad transcripts than zmubi1::aad transgenic plants. in tassel tissues, zmubi1::aad transgenic plants accumulate more aad transcript. these results demonstrate that the scbv-im promoter is stronger or comprable in strength to the strong, constitutively expressed maize ubiquitin promoter in young leaf and root tissues, but is weaker in the leaf below the devloping ear and in tassel tissues at r figure comparison of the scbv-im and zmubi promoters in transgenic maize tissues. transgenic, single copy maize events containing scbv-im::aad and zmubi1::aad constructs were analyzed for aad transcript accumulation in the youngest fully expanded leaf of v and v stage plants, the leaf just below the ear of r plants, a 1 cm section from the tip of the root in v and v stage plants, and in the tassels of r stage plants. three events for each of the two constructs were compared by rt-qpcr using primers specific to the aad transcript and normalized to an endogenous transcript, tip or maz. the error bars represent the standard deviation of three measurements. analysis of variance indicate that more aad transcript accumulate in the scbv-im::aad events than in zmubi1::aad events in leaves of v stage plants, the roots of v and v stage plants, while similar levels of aad transcript accumulate in leaves of v stage plants. zmubi1::aad transgenic plants accumulate more aad transcript than scbv-im::aad transgenic plants in leaf and tassel tissues at r scbv-im enhancer identification and characterization the activity of the scbv-im enhancer was demonstrated in transient assays first by identifying sequences that are necessary for high levels of transcription and then by identifying sequences that can enhance transcription from a heterologous promoter. the sequence of the scbv-im promoter is shown in figure 2; the transcription start site was mapped by 5′ race.figure scbv promoter sequence and fragments used in experiments. the transcription start site was mapped by 5′ race and sequences in the 5′ untranslated region of the transcript are underlined. the putative tata box is underlined at position − – - the scbv-im enhancer sequences are in bold. the fragments of the scbv-im promoter used in transient assays are displayed. the position of the transcription start site is shown. scbv-im promoter fragments scbv scbv and scbv were cloned upstream of the luciferase reporter gene. transcriptional activities of these constructs were tested by transfecting maize hi-ii suspension cells and monitoring relative activities of the reporter genes. to test the activities of different scbv-im promoter fragments, equal molar concentrations of the test plasmids and a reference plasmid, used as an internal control to normalize for differences in transformation efficiency, were co-introduced into maize hi-ii suspension cell cultures by particle bombardment. two days after the bombardment, total protein was isolated from transfected cells and luc and gus enzymatic activities were determined. activity was expressed as the ratio of luc to gus activity. the results show that the promoter fragment scbv had 60% of the activity of the scbv promoter fragment and the scbv promoter fragment had only 10% of the activity of the scbv fragment. these data indicate that sequences necessary for most of the scbv-im promoter activity reside upstream of the 333 bp fragment.figure promoter analysis of the scbv-im promoter. three fragments of the scbv-im promoter fused with the luciferase reporter gene and tested for activity in transient assays and compared with a luciferase construct without a promoter. these constructs were transfected into maize hi-ii cultures along with a zmubi1::gus construct. activity is reported as the ratio of luciferase activity from the test construct to gus activity from the internal control construct. error bars represent the standard deviation of three measurements. analysis of variance and tukey-kramer hsd tests indicate significant differences in lu/gu ratios for all constructs tested. next, two upstream fragments of the scbv-im promoter were tested for their ability to enhance transcription from a truncated heterologous promoter. two fragments of the scbv-im promoter were cloned upstream of a truncated maize alcohol dehydrogenase promoter fused to the firefly luciferase gene. these constructs were designated scbv282::zmadh1::luc and scbv537::zmadh1::luc, respectively. maize hi-ii suspension cells were transfected with plasmids scbv282::zmadh1::luc, scbv537::zmadh1::luc and zmadh1::luc along with the reference plasmid containing zmubi1::gus. overall, these promoters gave much lower activity than the intact scbv promoter. this may be because the zmadh chimeric promoters are inherently weaker than the intact scbv-im promoter or because a promoter fragment of the scbv-im promoter necessary for high activity was not included in the chimeric constructs.figure enhancer analysis of the scbv-im promoter. two fragments of the scbv-im promoter were fused with the truncated zmadh promoter and the luciferase reporter gene. these constructs were tested for activity in transient assays and compared with a construct containing only the minimal zmadh promoter. these constructs were transfected into maize hi-ii cultures with a zmubi1::gus construct. activity is reported as the ratio of luciferase activity from the test construct to gus activity from the internal control construct. error bars represent the standard deviation of three measurements. analysis of variance and tukey-kramer hsd tests indicate significant differences in lu/gu ratios for all constructs tested. the results shown in figure  indicate that the scbv fragment was able to enhance activity of the truncated zmadh promoter more effectively than the larger scbv fragment. these results indicate that these fragments of the scbv-im promoter cause an increase in activity of the reporter gene driven by a truncated heterologous promoter, and that most of this enhancing activity lies within the −434 bp to −153 bp region. to determine whether multiple copies of the scbv-im enhancer are more effective in activating transcription, and copies of the scbv fragment were cloned upstream of the truncated zmadh promoter fused to the luc gene and bombarded into maize hi-ii suspension cells. constructs containing and tandem copies of the scbv-im enhancer had times, times and times more activity, respectively, than did cells bombarded with the zmadh1::luc construct without any scbv-im sequences. it should be noted that in the experiment shown in figure  the activity of scbv282::zmadh1::luc and zmadh1::luc was substantially greater than in the experiment shown in figure  however, similar variation in promoter activity between independent experiments conducted with different cell preparations has previously been reported.figure analysis of scbv-im enhancer arrays. tandem copies of the scbv-im enhancer were fused with the truncated zmadh promoter and the luciferase reporter gene. these constructs were tested for activity in transient assays and compared with a construct containing only the minimal zmadh promoter. these constructs were transfected into maize hi-ii cultures with a zmubi1::gus construct. activity is reported as the ratio of luciferase activity from the test construct to gus activity from the internal control construct. error bars represent the standard deviation of three measurements. analysis of variance and tukey-kramer hsd tests indicate significant differences in lu/gu ratios for all constructs tested. scbv-enhancer activity in stable maize transformants to determine whether the scbv-im enhancer can increase expression of genes within the maize genome, an activation tagging construct consisting of the 4x tandem array of the scbv enhancer and a selectable marker composed of the rice actin promoter driving expression of the aad herbicide resistance gene and the maize lipase 3′ utr was cloned and transformed into maize plants via agrobacterium-mediated transformation.figure schematic of enhancer construct, peps used in maize transformation. the construct includes an array of copies of the scbv-im enhancers and the aad herbicide resistance gene containing the rice actin promoter fused with the aad gene and the maize lipase 3′ utr relative to the left border and right border of agrobacterium t-dna. transformants were examined for the location of the t-dna insertion and the proximity of the enhancers to annotated genes in the maize genome that were reported to be expressed at moderate levels in leaf tissues. determination of the site of integration of the construct was attempted for events by a transgene border sequence identification method and of these events were mapped to locations in the maize b reference genome. to determine whether the enhancers within the t-dna sequence are able to cause an increase in transcript accumulation of endogenous genes, these transformants were examined to identify t-dna insertion sites within ~ kb of a gene. the camv 35s enhancer has been demonstrated to up-regulate genes within ~ kb of the enhancer sequences. eleven events were examined to determine whether transcripts of genes adjacent to the activation tagging element were more abundant in the transgenic events than non-transgenic control plants. for five of these genes, no transcripts were detected in either the transgenic or non-transgenic lines. for the six other genes, transcripts were detected from genes adjacent to the activation tagging element. in these events, two genes showed a similar level of these transcripts in transgenic and non-transgenic plants. however, in other events the transcripts of genes adjacent to the activation tagging element were more abundant in the transgenic plant than the non-transgenic plant. for two genes, the transgenic plant showed more transcript than did the non-transgenic plant; this increase in abundance was and fold, respectively. for the other two genes, no transcript was detectable in the non-transgenic plant but transcript was clearly detectable in the transgenic plant. these results demonstrate the 4x tandem array of the scbv-im enhancer can increase transcript abundance in a stable transformed maize plant and in some cases may cause ectopic expression of genes that are not expressed, or expressed at very low levels. it also indicates that the 4x tandem array of the scbv-im enhancer meets the traditional definition of an enhancer because it can function upstream or downstream of the transcription unit and in either orientation.figure analysis of enhancer activity in transgenic maize plants. the relative locations of the activation tagging element and the gene examined are shown. the activation tagging element is represented by four yellow boxes representing the 4× scbv enhancer tandem array and the blue arrow representing the aad selectable marker cassette. maize genes are represented by the green box with the transcription start site and direction represented by the arrow. distance to promoter reflects the estimated distance from the enhancers to the translational start site of the gene. the relative transcript abundance is the amount of transcript of the gene in transgenic plants compared with the level of the transcript in non-transgenic plants. an asterix indicates that the values were significantly different as determined by a t-test. discussion the promoter sequences that we define include significant portions of the scbv orf iii gene. the scbv sequence, which has the greatest activity in transient assays, overlaps with protein coding nucleotides. the sequences overlapping the orf iii gene contain most of the promoter activity as demonstrated by the scbv fragment containing only 20 bp of the orf iii gene and having just 10% of the promoter activity of the scbv sequence. the scbv enhancer fragment contains 189 bp of orf iii coding sequence. a similar situation is found in arabidopsis where regulatory elements for the promoter of zwichel gene are found in exon and intron sequences of the adjacent hydroxyisobutyrl-coa hydrolase gene. the enhancer sequences in the scbv-im promoter were able to increase the activity of the truncated zmadh promoter, but these chimeric promoters were much weaker than the intact scbv-im promoter in the transient assays. this difference is so great it is unlikely to be the result of different cell preparations and may be the result of the scbv-im upstream activating sequences interacting differently with the heterologous core zmadh promoter and the native scbv-im promoter. similar results were seen when the camv 35s enhancer was placed upstream of the camv 19s core promoter. deletion analysis of the scbv-im promoter showed that removing sequences from − to − caused a 30% decline in promoter activity, while removing sequences from − to − caused a 90% decline in activity. it was, therefore, somewhat surprising to observe that the chimeric promoter scbv537::zmadh had less activity than the chimeric promoter scbv282::zmadh since the longer fragment in the promoter deletion analysis had the most activity. surprising results are often obtained when portions of promoters are added or deleted and even small portions of promoters can have dramatic effects. for example, dey and matti showed that removing 50 bp of the mmv promoter increased activity fold and simon et al. showed that deleting 54 bp of the inner no outer promoter of arabidopsis could reverse a silenced promoter. multiple copies of the scbv-im enhancer cause an increase in activity of the chimeric promoters in transient assays. this is consistent with what has been observed with the camv 35s and fmv ehancers and may be due to multiple copies of the enhancer being more efficent in recruiting transcription factors to the promoter. the scbv fragment is capable of acting as a transcriptional enhancer when present in the maize genome in a 4x tandem array. in events containing the 4x scbv-im enhancer upstream and downstream of genes, and in either orientation with respect to these genes, increased transcript accumulation was observed. furthermore, the element appeared to cause accumulation of transcripts that are not present, or present in very low levels, in non-transgenic lines. this demonstrates that the scbv-im enhancer may be used for activation tagging in maize. the scbv-im enhancers increased expression of out of genes that showed non-detectable expression in non-transgenic control plants. this is similar some studies that have reported ectopic expression of genes when the camv 35s enhancers integrate nearby.  in this work, we demonstrate that the scbv-im promoter is comparable in strength to the zmubi promoter in transgenic young maize leaves and roots and we identify sequences from the scbv-im promoter that can function as a transcriptional enhancer in maize plants. we used transient assays to identify promoter sequences that are responsible for most of the promoter activity and sequences of this promoter that enhance expression from a heterologous promoter. finally, we generated stable transgenic plants containing 4x tandem arrays of the scbv-im enhancer and demonstrated that transcripts of genes near the insertion site are more abundant than in non-transgenic control plants. activation tagging by randomly inserting transcriptional enhancers in the genome is a powerful tool for identifying gene function. the camv 35s enhancer has been used to develop activation tagging systems for arabidopsis, rice and barley. using these activation tagging systems, researchers have identified a number of genes with novel functions. to date, no activation tagging system has been developed for maize. as a first step in developing an activation tagging system for maize, we have identified a transcriptional enhancer from scbv-im and have shown it to be able to activate transcription from a truncated zmadh promoter in transient assays and from endogenous promoters in transformed maize plants. 
in transgenic maize, the scbv-im promoter was shown to be comparable in strength to the maize ubiquitin promoter in young leaf and root tissues. the scbv-im enhancer can activate transcription upstream or downstream of genes and in either orientation. enhancer sequences were identified and shown to increase the activity of a heterologous truncated promoter. these enhancer sequences were shown to be more active when arrayed in copy arrays than in or copy arrays. transcriptional enhancers have been used to enhance expression of specific promoters in transgenic plants and in activation tagging studies to help elucidate gene function. a transcriptional enhancer from the sugarcane bacilliform virus - ireng maleng isolate that can cause increased transcription when integrated into the the genome near maize genes has been identified. transcriptional enhancers are able to increase transcription from heterologous promoters when placed upstream, downstream and in either orientation, relative to the promoter. the promoter was dissected to identify sequences that confer high activity in transient assays. when the enhancer array was transformed into maize plants it caused an increase in accumulation of transcripts of genes near the site of integration in the genome.
 transcriptional enhancers are able to increase transcription from heterologous promoters when placed upstream, downstream and in either orientation, relative to the promoter. transcriptional enhancers have been used to enhance expression of specific promoters in transgenic plants and in activation tagging studies to help elucidate gene function.  a transcriptional enhancer from the sugarcane bacilliform virus - ireng maleng isolate that can cause increased transcription when integrated into the the genome near maize genes has been identified. in transgenic maize, the scbv-im promoter was shown to be comparable in strength to the maize ubiquitin promoter in young leaf and root tissues. the promoter was dissected to identify sequences that confer high activity in transient assays. enhancer sequences were identified and shown to increase the activity of a heterologous truncated promoter. these enhancer sequences were shown to be more active when arrayed in copy arrays than in or copy arrays. when the enhancer array was transformed into maize plants it caused an increase in accumulation of transcripts of genes near the site of integration in the genome.  the scbv-im enhancer can activate transcription upstream or downstream of genes and in either orientation. it may be a useful tool to activate enhance from specific promoters or in activation tagging. keywords promoterenhancertranscriptiontransgenic planttransient assayissue-copyright-statement© the author 2014 