Analysis of the SHP 2 enhancer for the use of tissue specific activation tagging in Arabidopsis thaliana

Activation tagging is a powerful tool to identify new mutants and to obtain information about possible biological functions of the overexpressed genes. The quadruple cauliflower mosaic virus (CaMV) 35S enhancer fragment is a strong enhancer, which is most commonly used for this purpose. However, the constitutive nature of this enhancer may generate lethal mutations or aberrations in different plant organs by the same overexpressed gene. A tissue-specific activation tagging approach may overcome these drawbacks and may also lead more efficiently to the desired phenotype. For this reason the SHATTERPROOF2 (SHP2) promoter fragment was analysed for enhancer activity. The SHP2 gene is involved in dehiscence zone development and expressed during silique development. The aim of the experiments described here was to identify a dehiscence zone specific enhancer that could be used for tissue-specific activation tagging. The chosen SHP2 enhancer fragment was found to be expressed predominantly in the dehiscence zone and showed enhancer activity as well as ectopic expression activity. This activity was not influenced by its orientation towards the promoter and it was still functional at the largest tested distance of 2.0 kb. Based on these results, the SHP2 enhancer fragment can potentially be used in a tissue-specific activation tagging approach to identify new Arabidopsis mutants with an altered dehiscence zone formation.


Introduction
Activation tagging has become an upcoming tool to generate mutant plants.It is an alternative approach for gene function analysis, because loss-of-function mutations has its limitation in cases of functional gene redundancy (Arabidopsis Genome Initiative, 2000).Activation tagging was proposed as a novel gene isolation method in which a gene is either ectopically or constitutively overexpressed compared to normal expression levels (Walden et al., 1994).Walden et al. (1994) designed a T-DNA based activation tagging approach to identify and isolate novel genes from tobacco, and since then it has been largely applied using either T-DNA insertion strategies (Borevitz et al., 2000;Ito and Meyerowitz, 2000;Lee et al., 2000;van der Graaff et al., 2000;Weigel et al., 2000;Huang et al., 2001) or transposon based approaches (Wilson et al., 1996;Marsch-Martinez et al., 2002).This technology has been applied successfully to many plant species like Arabidopsis, rice, tomato, petunia and tobacco (Weigel et al., 2000;Jeong et al., 2002;Zubko et al., 2002;Ahad et al., 2003;Mathews et al., 2003).
Activation tagging is based on strong transcriptional enhancer sequences that can activate gene expression in the vicinity of the site where the enhancer was inserted into the genome.The most commonly used enhancer is a quadruple combination of the cauliflower mosaic virus (CaMV) 35S enhancer (Odell et al., 1985;Hayashi et al., 1992).These 4x35S enhancer elements have been reported to strongly enhance endogenous gene expression rather than ectopically or constitutively overexpress genes (Neff et al., 1999;van der Graaff et al., 2000).It has been demonstrated that it can stimulate gene expression of neighboring genes independently of its orientation, up to a distance of 3.6 kb (Weigel et al., 2000) or even up to 5.0 kb (Chalfun-Junior et al., 2005).
It is tempting to use this technique in a random-like approach to isolate new mutants and analyse in more detail the overexpressed genes.In some cases, the mutant displays a phenotype that can either be directly associated with the gene function of the activated gene (Zubko et al., 2002) or may provide an indication of the pathway in which the gene is involved (Kardailsky et al., 1999;Zhao et al., 2001;Yuen et al., 2003).The activation tagging method has also been used as a novel approach to isolate suppressor mutants of known mutant phenotypes (Neff et al., 1999;van der Graaff et al., 2003).However, when searching for specific mutants with a more specific phenotype, the 4x35S enhancer is not very attractive to use, as it will induce aberrations in plant tissues other than the specific one that was intended to be modified.In that case, tissue-specific enhancement could directly lead to the mutants of interest.To test this hypothesis we have characterized a tissue-specific enhancer that may be applicable to efficiently generate activation mutants with an altered pod shattering phenotype.
Precocious pod shattering is a phenomenon in which fruits lose their seeds before harvesting time, generating high losses of agronomic crops, e.g., rapeseed.Engineering for shatter resistant plants allow the plants to grow until the fruits are fully ripened, resulting in an optimal product quality and increased crop yield.The closely related MADS box transcription factors SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) control seed dispersal in Arabidopsis by regulating the development of the dehiscence zone (Ferrandiz et al., 2000;Liljegren et al., 2000;Ferrandiz, 2002).In shp1shp2 double mutant plants, the dehiscence zone is completely absent and the fruits fail to open.
Here, we describe the identification of a SHP2 enhancer taken from its natural promoter, which contains all necessary characteristics required for a tissue-specific activation approach.

Plant material and transformation
Constructs were introduced into Arabidopsis thaliana ecotype Col-0 using Agrobacterium tumenfaciens strain GV3101 and the floral dip method (Clough and Bent, 1998).For selection of transformants, seeds were surface sterilized by vapor phase sterilization (http://plantpath.wisc.edu/~afb/vapster.html), and selected on medium containing kanamycin.Plants were grown in soil, under normal greenhouse conditions (22 °C, 14/10 h light/dark).

Constructs
The SHP2 enhancer fragment Primers were designed at -1275 bp and -55 bp from the transcription initiation site of the SHP2 gene (forward primer SHP2F 5'-GTCGACAAGCTTAAGTTCTTTCTT GAAATG-3', reverse primer SHP2R 5'-GTCGACAA GCTTCACTTAACTGCTGCTTCAAC-3').Both primers were extended with sequences for digestion sites to facilitate subcloning (HindIII and SalI sites underlined).DNA of Arabidopsis thaliana ecotype Col-0 was used as template for PCR amplification.Using these primers a band of the expected 1220 bp was obtained, cloned (in pGEMT-Easy®, Promega) and sequenced to confirm its identity.

SHP2 enhancer fused to minimal -47-35S::GUS
The minimal -47-35S promoter GUS vector, GUSXX-47 (Pasquali et al., 1994) was obtained from J. Memelink, (Leiden University, Leiden, The Netherlands).The SHP2F primer was extended with a HindIII site and the reverse primer with a SalI site, the fragment amplified and ligated in the HindIII-SalI site upstream of the -47-35S promoter, which was fused to the GUS reporter gene.The whole fragment was subcloned in pBINPLUS (van Engelen et al., 1995) using HindIII-KpnI, resulting in vector pGD751.
SHP2 fragment fused to the pFBP1::GUS Both SHP2 primers were extended with both HindIII and SalI sites to facilitate the cloning of the vectors described next.Several constructs were generated to test the enhancer activity of the SHP2 fragment.For that reason the SHP2 fragment (as a HindIII-HindIII fragment) was ligated upstream of the FBP1 promoter present in vector pFBP12E (Angenent et al., 1993).This resulted in a sense fusion of the SHP2 enhancer to the 'short' 220 bp pFBP1::GUS (pGD418).
The SalI-SalI fragment was introduced in the SalI digested pFBP12E vector, resulting in a sense fusion of the SHP2 enhancer to the 'long' 1040 bp FBP1 promoter fused to the GUS reporter gene (pGD393).For the other constructs, the SalI-SalI fragment of the cloned PCR SHP2 enhancer fragment was first subcloned in pBluescript SK + vector (Promega) in both orientations (pARC012 and pARC013).The 'long' pFBP1::GUS fragment was also subcloned as an EcoRI-EcoRI from pFBP12E into pBluescript SK + vector in both orientations (pARC014 and pARC015).A ClaI-XhoI fragment containing the sense or antisense SHP2 enhancer was then inserted adjacent to the pFBP1::GUS fragment in both vectors pARC014 and pARC015.This resulted in an antisense fusion of the SHP2 enhancer upstream of the 'long' pFBP1::GUS fragment and in vectors where the SHP2 enhancer was downstream of this fragment in either sense or antisense orientation.The total inserts of these vectors were subcloned in the binary vector pBINPLUS® by using XbaI and KpnI.All vectors were checked in detail by restriction sites and fragment length analysis.

GUS activity
Histochemical localization of GUS activity was performed as described by Jefferson et al. (1987).

Tissue specificity of the SHP2 enhancer
It has been shown previously that the SHP2 gene is expressed in the dehiscence zone of Arabidopsis siliques (Savidge et al., 1995;Liljegren et al., 1998;Liljegren et al., 2000).To test whether a putative SHP2 enhancer fragment contains the cis-acting elements and all its tissue specific regulating sequences, a 1220 bp upstream sequence was tested for its promoter/enhancer activity.This 1220 bp fragment was chosen from -1275 bp to -55 bp upstream of the transcription initiation site, which was also 8 bp upstream of a putative TATA box.This 1220 bp SHP2 promoter fragment contains a CarG-box like sequence (Savidge et al., 1995), which is the putative AGAMOUS binding site and which is very likely to be important for its regulation.This promoter fragment (from now on referred to as the SHP2 enhancer) was fused to a minimal -47-35S promoter, which was fused to the GUS reporter gene (GUSXX-47) (Pasquali et al., 1994).This construct will allow the analysis of its transcriptional enhancer activity and tissue specificity.This binary vector (Figure 1A) and a control construct, which carries only the empty vector (GUSXX-47), were introduced into Arabidopsis plants ecotype Col-0.In Table 1, the GUS expression patterns of 24 analyzed T1 plants are summarized.No GUS expression was observed in plants containing only the empty vector.In most of the GUS positive plants, GUS staining was observed in the dehiscence zone (Figure 1B).This expression in the dehiscence zone was seen at earlier stages during flower development (Figure 1C).Besides GUS expression in the dehiscence zone, some plants also displayed GUS expression in other tissues, for instance in pollen grains (Figure 1D), ovules (Figure 1E), funiculus (Figure 1F), nectaries (Figure 1G) and the vascular junction at the receptacle (Figure 1H).Similar to the results obtained here, SHP1 and SHP2 genes were previously described to be expressed in tissues like septum, ovules and funiculus (Ma et al., 1991), in addition to the dehiscence zone (Savidge et al., 1995;Liljegren et al., 1998;Liljegren et al., 2000).In some plants, no GUS staining was observed.Although no molecular analyses were performed on them, it is very likely that these plants were not transgenic or were not expressing the GUS gene properly (Table 1).Based on these results, it can be concluded that the 1220 bp SHP2 enhancer still contains the dehiscence specific regulatory sequences of the SHP2 promoter.

Activity of the SHP2 enhancer
Enhancers are defined as cis-acting DNA sequences that can increase the transcription level of genes by the binding of specific transcription factors.Full promoter activity also requires the DNA region containing the TATA box directly upstream of the transcription start site (Lewin, 2000).Enhancers usually can function in either orientation and separated from a minimal promoter domain.To obtain an enhancer for efficient tissue-specific activation tagging, these features are essential.To test all these features of the SHP2 enhancer, several constructs were generated (Figure 2).
Because enhancers are not able to act alone (Lewin, 2000), a minimal or natural promoter is necessary.Therefore, the FLORAL BINDING PROTEIN1 (FBP1) promoter was chosen because this petunia promoter regulates expression in whorl 2 and 3 of the flower (petals and stamens respectively) (Angenent et al., 1993).In petunia, two different lengths of the FBP1 promoter fragment were analysed, a 220 bp 'short' promoter and a 1040 bp 'long' FBP1 promoter.Both promoter fragments showed the same specificity and levels of expression in petunia (Angenent et al., 1993).Analysis of both promoter fragments in Arabidopsis showed the same results as for petunia (unpublished re-SHP2 enhancer for the use of activation tagging 403 sults).Therefore, as control, a binary vector harbouring the 'long' FBP1 promoter sequence fused to GUS was introduced into Arabidopsis plants.Several transgenic plants were obtained with different levels of GUS expression in petals (often very weak) and filaments (stronger) (Table 2 and Figure 3A, B).This result indicates that this promoter acts as expected, with the same specificity as in petunia (Angenent et al., 1993), and can be used to test the SHP2 enhancer.Figure 2 summarises the vectors that were used to test the SHP2 enhancer activity based on its ectopic expression in combination with the FBP1 promoter.In the first two tester constructs, the SHP2 enhancer was fused in sense orientation, with both the 'short' and the 'long' versions of the FBP1 promoter resulting in pGD418 and pGD393 respectively (Figure 2).These constructs were used to test whether the SHP2 enhancer fragment could ectopically activate GUS expression in combination with the FBP1 promoter and, moreover, whether the enhancer still keeps its tissue-specific activity when combined with a natural promoter.By using the 'short' and 'long' version of the FBP1 promoter, the influence of the distance towards the promoter was analysed.The other three constructs should identify whether the SHP2 enhancer acts independently of its orientation and position related to the coding sequence.The results of the GUS staining of the transgenic Arabidopsis plants expressing these constructs are summarised in Table 2.
In general, all constructs that contained the SHP2 enhancer fused in either way to the pFBP1::GUS, showed the FBP1-driven expression in whorls 2 and 3 (Figure 3C) and, also ectopic GUS expression in the dehiscence zone (Figure 3D).In whorls 2 and 3, the GUS expression driven by the FBP1 promoter was similar to or in some lines slightly enhanced compared to the expression observed in plants that contained the control construct with only the 'long' pFBP1::GUS construct (pARC069).The SHP2 enhancer region drove ectopic expression to the dehiscence zone, however it did not seem to promote up-regulation of the FBP1 promoter activity.As expected, this ectopic expression driven by the SHP2 enhancer was mostly seen in the dehiscence zone tissue.However in some plants, GUS staining in pollen grains was observed as well (data not shown).
The distance of the SHP2 enhancer relative to the promoter sequence did not seem to influence the frequency of plants showing ectopic expression in the dehiscence zone.When the SHP2 enhancer was situated downstream of the GUS gene (pARC011 and pARC027) (Figure 2), the enhancer was still able to ectopically activate the expression with the same frequency as observed for the upstream sense position of the SHP2 enhancer (Table 2).
The only construct that did not result in ectopic expression in dehiscence zone tissue was construct pARC015 (Table 2), which contains the SHP2 enhancer positioned upstream, in an anti-sense orientation, linked to the 'long' pFBP1::GUS fragment.Out of fourteen T1 plants analysed, 10 showed expression in whorls 2 and 3 only.

Discussion
Tissue specific activation tagging is a novel approach to generate mutants for a specific trait of interest.Requirements for such a strategy are that: i) the used enhancer frag-404 Chalfun-Junior et al.   ment shows tissue specificity, avoiding unwanted side effects in other tissues ii) the activity of the enhancer should still be functional at least at several kbs from the natural promoter or gene, increasing the efficiency that genes will be activated, and iii) the insertion in the genome should be carried out by a system that minimizes the chances of complex insertion integrations (Nacry et al., 1998), which can cause silencing of the introduced enhancer (Chalfun-Junior et al., 2003).
Here we show that the 1220 bp SHP2 promoter fragment taken from the upstream sequence of the SHP2 gene contains most of the cis-regulatory sequences.It has been previously reported that the SHP2 gene is expressed in the dehiscence zone of the Arabidopsis silique, in addition to other tissues like septum, ovules and funiculus (Ma et al., 1991;Savidge et al., 1995;Liljegren et al., 1998;Liljegren et al., 2000).The SHP2 enhancer sequence chosen in our study is mainly expressed in the dehiscence zone, although GUS expression was also observed in ovules, funiculi and nectaries.The expression occasionally observed in a few pollen grains, nectaries and the vascular junction at the receptacle were not described previously.We did not test GUS expression driven by the complete endogenous SHP2 promoter in this same setup, which makes a comparison between published in situ hybridisation data and our data obtained with the enhancer studies more difficult.In conclusion, we demonstrated that it is possible to confer specific and ectopic expression of a particular gene by using an upstream enhancer sequence combined with minimal promoter elements.
The second prerequisite for an efficient activation tagging approach is that the enhancer activity should reach as far as possible from the minimal promoter elements (e.g., the TATA box).Based on the results obtained with the constructs in which the SHP2 enhancer was fused to the FBP1 promoter (in combination with the GUS reporter gene), we detected ectopic expression driven by the SHP2 enhancer in most of the combinations.In some of the plants, the GUS staining in the dehiscence zone was very strong and comparable with -47-35S::GUS transgenics.This was even the case when the enhancer was inserted downstream of the GUS gene, which resulted in GUS expression levels comparable with those for the upstream sense positions.
It still remains unclear why the construct pARC015 revealed no ectopic activation.This improper function may be specific for the FBP1 promoter and it is unlikely that this SHP2 enhancer for the use of activation tagging 405 is a general feature for an enhancer inserted in antisense orientation upstream of a native promoter.Our results demonstrate that the enhancer activity acts over a distance of at least 2.0 kb, making it interesting to explore it in a tissue-specific activation tagging strategy.When an even stronger activity would be required, it may be possible to create a tagging construct with a repeated SHP2 enhancer, similar to experiments performed with the 35S enhancer (Weigel et al., 2000;Marsch-Martinez et al., 2002).And this brings us to the third requirement for an efficient tagging strategy, i.e. avoidance of silencing, which could be induced by the insertion of repeated DNA sequences (Chalfun-Junior et al., 2003).
Based on the findings described by Marsch-Martinez et al. (2002) and our own observations on the methylation of T-DNA based activation tagging using the quadruple 35S enhancer (Chalfun-Junior et al., 2003), we propose a transposon-based tagging strategy as being the most promising.Hopefully this strategy, in which the described SHP2 enhancer could be used as tissue-specific enhancer, will lead to a better understanding of pod dehiscence and to novel approaches to control the shattering behaviour of crops.

Figure 1 -
Figure 1 -GUS expression pattern in Arabidopsis plants containing the construct SHP2 fused to the minimal 35S promoter (-47-35S::GUS).(A) Schematic representation of the construct (which is not drawn to scale).(B) Arabidopsis silique showing GUS expression in the dehiscence zone (DZ, arrow).(C) A young flower with GUS expression in the DZ.GUS expression is also present in (D) pollen grains, (E) ovules, (F) funiculus and (G) nectary (ne), and (H) vascular junction (vj) in the receptacle.

Figure 2 -
Figure 2 -Schematic representation of the constructs in which the SHP2 enhancer was fused to the pFBP1::GUS promoter-gene construct in different orientations.pARC069: the pFBP1::GUS construct used as control.pGD418: SHP2 in sense orientation upstream of the short version of the pFBP1 promoter.pGD393: the SHP2 enhancer fragment is located upstream of the long pFBP1::GUS version.pARC015: the SHP2 is also located upstream of the long pFBP1::GUS construct, however in antisense orientation.pARC011: the SHP2 is placed downstream of the GUS gene in sense orientation and SHP2 is placed in antisense orientation in the same long version of the FBP1 promoter, in pARC027.This schematic representation is not drawn to scale.

Table 2 -
GUS expression observed in tissues of transgenic Arabidopsis plants containing different constructs (see Figure2).