Planta (2005) 221: 523–530 DOI 10.1007/s00425-004-1466-4
O R I GI N A L A R T IC L E
So Yeon Yoo Æ Kirsten Bomblies Seung Kwan Yoo Æ Jung Won Yang Æ Mi Suk Choi Jong Seob Lee Æ Detlef Weigel Æ Ji Hoon Ahn
The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene
Received: 21 October 2004 / Accepted: 23 November 2004 / Published online: 29 January 2005 Springer-Verlag 2005
Abstract Positive selection of transgenic plants is essential during plant transformation. Thus, strong promoters are often used in selectable marker genes to ensure successful selection. Many plant transformation vectors, including pPZP family vectors, use the 35S promoter as a regulatory sequence for their selectable marker genes. We found that the 35S promoter used in a selectable marker gene affected the expression pattern of a transgene, possibly leading to a misinterpretation of the result obtained from transgenic plants. It is likely that the 35S enhancer sequence in the 35S promoter is responsible for the interference, as in the activation tagging screen. This affected expression mostly disappeared in transgenic plants generated using vectors without the 35S sequences within their T-DNA region. Therefore, we suggest that caution should be used in selecting a plant transformation vector and in the interpretation of the results obtained from transgenic approaches using vectors carrying the 35S promoter sequences within their T-DNA regions. S. Y. Yoo Æ S. K. Yoo Æ J. W. Yang Æ M. S. Choi Æ J. H. Ahn (&) Plant Signaling Network Research Center, School of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Korea E-mail:
[email protected] Tel.: +82-2-3290-3451 Fax: +82-2-927-9028 J. S. Lee School of Biological Sciences, Seoul National University, Seoul, 152-742, Korea D. Weigel Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tu¨bingen, Germany K. Bomblies Æ D. Weigel Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA School of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Korea
Keywords 35S enhancer Æ 35S promoter Æ Misexpression Æ Plant transformation vector Æ Trans activation Abbreviations aacC1: Gentamycin acetyltransferase Æ aadA: Streptomycin/spectinomycin adenylyltransferase Æ mas: Mannopine synthase Æ nptII: Neomycin phosphotransferase
Introduction The introduction of a gene-of-interest into a plant cell is indispensable for plant biologists to study the function of the gene and its underlying molecular mechanism in plants. During plant transformation experiments, positive selection of transgenic plants from a non-transgenic background is prerequisite to the generation of stable transgenic plants (Angenon et al. 1994). A large number of positive selectable marker genes have thus been developed, such as antibiotics, antimetabolite and herbicide resistance genes (Miki and McHugh 2004). An efficient selection system is required because the choice of selectable marker genes depends on the plant species and the specific genotype of the plant. Therefore, strong promoters are frequently used to ensure abundant transcription of the selectable marker genes. One of the commonly used strong promoters in selectable marker genes is the 35S promoter sequence from the Cauliflower mosaic virus (CaMV) (Franck et al. 1980). The 35S promoter effectively puts its downstream gene outside virtually any regulatory control by the host genome and expresses the gene at approximately two to three orders of magnitude higher, thus allowing a strong positive selection. A number of plant transformation vectors, including pPZP family vectors, the pCAMBIA series and pINDEX1, use the 35S promoter in their selectable marker genes (Hajdukiewicz et al. 1994; Ouwerkerk et al. 2001). These
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vectors have been widely used as carriers of genes-ofinterest or for high throughput analysis (Curtis and Grossniklaus 2003; Fang and Fernandez 2002; Jeong et al. 1999; Kang et al. 2003; Zhao et al. 2002). We also generated a group of constructs using pPZP family vectors, and introduced them into plants. Interestingly, we found that the expression of a transgene subcloned into pPZP family vectors was affected; both tissue localization patterns and expression levels of the transgene were significantly altered. However, this interference mostly disappeared in transgenic plants generated using a plant transformation vector without the 35S promoter. One example was a promoter fusion construct of FT (Flowering Locus T) (Kardailsky et al. 1999). FT acts as a floral inducer, and its RNA preferentially accumulates in the above-ground parts, especially in vascular tissues (Kardailsky et al. 1999; Takada and Goto 2003). To test for misexpression, we fused FT cDNA with the promoter of LRP1 (Lateral Root Primordia1), a root-specific gene (Smith and Fedoroff 1995). The resulting LRP1::FT chimeric gene introduced into plants using pPZP212 caused precocious flowering, with LRP1::FT RNA strongly expressed in all tissues of the transgenic plants. In contrast, when the same LRP1::FT was introduced using pCGN1547 (McBride and Summerfelt 1990), which does not carry the 35S promoter, the flowering phenotype of the transgenic plants was largely normal, and the strong expression of LRP1::FT disappeared. Another experiment using the UFO::GUS construct also showed different expression patterns depending on the plant transformation vectors. In this study, we have shown that the expression of a transgene subcloned into a plant transformation vector containing the 35S promoter sequence in its selectable marker gene was affected, probably due to its 35S enhancer sequences (Benfey et al. 1990a; Benfey et al. 1990b). This suggests that the 35S enhancer sequences in the T-DNA region of a plant transformation vector trans activate and override the control of the transgene expression, as did the 35S enhancers in the activation tagging screen (Weigel et al. 2000). Therefore, we suggest that the plant transformation vectors carrying the 35S promoter in a selectable marker gene should be avoided during transformation to prevent misinterpretation of the subsequent results.
Materials and methods Plant materials and measurement of flowering time Wild-type Columbia plants were used for plant transformation and were grown in Sunshine Mix 5 (Sungro Horticulture, Quincy, MI) under long day conditions (16 h:8 h light:dark) at 23C. A modified floral dip method was used to introduce recombinant plasmids into Arabidopsis (Clough and Bent 1998). Transgenic seedlings were selected on solid MS media, containing 50 lg/ml kanamycin or 100 lg/ml gentamycin, and
transferred to soil. The flowering times of the transgenic plants carrying pJA1022 and pJA1102 plasmids were measured by counting the total leaves of at least 13 plants in the T1 generation. Construction of recombinant plasmids To construct an LRP1::FT construct, an LRP1 promoter was amplified from genomic DNA of wild-type Columbia plants with a pair of primers, JH1038 (5¢-AT GGTACCACGT GTGCCATATTAATAACGAAGTAT-3¢) and JH1039 (5¢-TA CCCGGGAGCTCTCCG TCGCCGCCGCCGAAG-3¢), where the synthetic restriction sites are underlined. The LRP1 promoter was fused with the FT cDNA and nos terminator, resulting in a pJA1018 clone. A restriction fragment obtained from the digestion of the pJA1018 plasmid with KpnI and HindIII was subcloned into pPZP212 and pCGN1547. The resulting plasmids were named pJA1022 and pJA1102, respectively, and were introduced into Arabidopsis plants. To generate UFO::GUS, a UFO promoter, 4.1 kb in length, was isolated from a BAC clone and fused to a GUS reporter gene. The UFO::GUS gene was subcloned into pPZP222, and the resulting recombinant plasmid named KB68. To change its vector backbone, an EcoRI fragment carrying the UFO::GUS gene was isolated from a KB68 clone, and subcloned into pJHA212B, generating a pSYY003 clone. To construct pJHA212K, pJHA212G, and pJHA212B, nptII, aacC1 and BAR genes were amplified from pPZP212, pPZP222, and SKI015 using gene-specific primers. An nptII gene was amplified from pPZP212 with the primers, JH1149 (5¢-GGGAAGCTG ACTAGTCGGGGTGGG-3¢) and JH1150 (5¢-TAGCGAATT ACTAGTATCGTTTCG-3¢). A pair of primers, JH1151 (5¢-TCGATCGAC ACTAGTTAGAACGAA3¢) and JH1152 (5¢-GATTCGCAG ACTAGTCGATCGACC-3¢), was used to amplify the aacC1 from pPZP222. Another pair of primers, JH1153 (5¢-ATCGATCCC ACTAGTCATCACATC-3¢) and JH1154 (5¢-AATTACTATTT ACTAGTACCATGAGCC-3¢), was used to amplify the BAR gene from SKI015. The underlined sequences in these oligonucleotides are synthetic SpeI sites. The amplified products were subcloned between the mas promoter and terminator sequences derived from pCGN1547 to make selectable marker gene cassettes. The cassettes were ligated with a SacI/ Eco0109I fragment of pPZP212 not containing an nptII gene. Reverse-transcriptase PCR The reverse transcriptase-mediated PCR procedure has been described previously (Kardailsky et al. 1999). After DNaseI treatment, complementary DNA was synthesized from the total RNA isolated from various tissues
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of pJA1022 and pJA1102 plants. A pair of primers, JH1093 (5¢-CTTCTGTCCGTTACGGCCTAC-3¢) and JH1065 (5¢-CTAAAGTCTTCTTCCTCCGCA-3¢), was designed to specifically amplify the LRP1::FT transgene from the cDNA. JH1011 (5¢-GATCTTTGCCGGAA AACAATTGGAGGATGGT-3¢) and JH1012 (5¢-CG ACTTGTCATTAGAAAGAAAGAGATAACAGG-3¢) primers were used to amplify the UBQ10 used for a positive control. Histochemical GUS assay and microscopy GUS staining and microscopic sections of the shoot apical regions were performed as described previously (Sessions et al. 1999). GUS staining in primary inflorescences was photographed using a Nikon SMZ1000 dissection microscope (Tokyo, Japan). Stained primary inflorescences were fixed and embedded in paraffin. After deparaffinization, the sections were examined under dark field conditions using a Nikon Optiphot-2 microscope (Tokyo, Japan).
Results and discussion Plant transformation vectors with or without the 35S sequences led to different results To understand the role of FT, which is expressed in the above-ground parts of Arabidopsis seedlings (Kardailsky et al. 1999), the FT expression was directed to the underground parts by using a root-specific promoter. We fused FT cDNA to the LRP1 promoter (Smith and Fedoroff 1995) and subcloned the resulting chimeric gene into pPZP212, and named the recombinant plasmid pJA1022. The pJA1022 clone was introduced into Arabidopsis and the effect of FT expression tested in the roots of the transgenic plants. pJA1022 plants showed moderately precocious flowering under long days (Fig. 1a). The transgenic plants flowered with 7.2 leaves, whereas wildtype Columbia and SKI083 plants, the reference line constitutively expressing FT, flowered with 14.3 leaves and 4.5 leaves, respectively, under the same conditions. All pJA1022 plants showed early flowering, displaying a narrow deviation of leaf numbers, indicating that LRP1::FT acted similar to 35S::FT. Although LRP1::FT in pJA1022 plants accelerated flowering, pJA1022 plants showed no determinate inflorescences (Fig. 1b), suggesting that the FT expression driven by the LRP1 promoter affected only the phase transition of transgenic plants. The observation that root-specific expression of FT accelerated flowering raised the possibility of long-distance transmission of floral induction signals being generated somewhere other than the shoot apical meristem, which coincided with the florigen concept (Aukerman and Amasino 1998; Colasanti and Sundaresan 2000). To confirm that the root-specific expression of FT accelerated flowering, another commonly used binary
Fig. 1 a Distributions of the total number of leaves of SKI083, pJA1022, pJA1102 and wild-type Columbia plants during bolting under long day conditions. SKI083 plants showed early flowering, whereas pJA1022 plants showed moderately early flowering. In contrast, most pJA1102 plants flowered normally, as did wild-type Columbia plants, with the exception of a few lines. b Inflorescence morphologies of SKI083, pJA1022, pJA1102 and wild-type Columbia plants under long day conditions. SKI083 plants, in which FT was overexpressed, showed a determinate inflorescence after producing a few floral buds. However, pJA1022 and pJA1102 plants carrying LRP1::FT, showed no terminal flower phenotype. c Expression of the LRP1::FT transgene in pJA1022 and pJA1102 plants. The LRP1::FT transgene was ectopically detected in whole tissues of pJA1022 plants. TF Terminal flower
vector, pCGN1547 (McBride and Summerfelt 1990) was employed. We subcloned LRP1::FT into the vector, and named the resulting recombinant plasmid pJA1102. We introduced the pJA1102 plasmid into Arabidopsis and
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measured the flowering time of transgenic plants, as described above. The pJA1102 plants did not flower as early as those of pJA1022 (Fig. 1a). The average number of leaves of pJA1102 plants at bolting under long days was 11.2 leaves, with a wide standard deviation. This indicated that most pJA1102 plants flowered normally, as did wild-type Columbia plants, with the exception of a few lines, suggesting that root-specific expression of FT did not alter the flowering time. There are two possible explanations for this subset of plants showing early flowering. One is that this phenotypical variation resulted from a positional effect of the pJA1102 clone (Stam et al. 1997), such that T-DNA was integrated adjacent to an endogenous enhancer. An alternative possibility is that root-specific expression of FT may truly cause a phase change, reflecting the expression of a subset of flowering time genes, CO, FT and TFL1, in the vascular bundle of seedlings (Takada and Goto 2003). Nonetheless, the flowering time of the pJA1102 plants was significantly different (Student’s t-test; p < 0.0015) from that of pJA1022 plants, raising the question, why should the same LRP1::FT led to different results? To examine why the pJA1022 and pJA1102 plants showed different results, the expression pattern of the LRP1::FT transgene was investigated. We designed a set of primers specific to the LRP1 promoter and FT coding sequence, JH1093 and JH1065, respectively, so that LRP1::FT RNA was specifically detected. Total RNA was isolated from various tissues of pJA1022 and pJA1102 plants and reverse transcriptase-mediated PCR (RT-PCR) performed. LRP1:: FT RNA was unexpectedly detected in all tissues of pJA1022 plants (Fig. 1c); and furthermore, at high levels, suggesting transcriptional enhancement of LRP::FT. It was likely that this strong and ectopic expression of FT was responsible for the accelerated flowering of pJA1022 plants. However, in pJA1102 plants, LRP1::FT RNA messages were very low under the same PCR conditions (Fig. 1c), and in the early flowering lines LRP1::FT was expressed weakly in other tissues, which explains the moderate early flowering (data not shown). One possibility explaining this weak ectopic expression is the LRP1 promoter used in this study leaked slightly in tissues other than the roots. An alternative explanation could be that, in the early flowering lines of pJA1102 plants, the LRP1::FT transgene is inserted adjacent to an endogenous enhancer in the plant genome, as described above. The 35S enhancer sequences likely altered the expression of a transgene Because only the transgene and a selectable marker gene were integrated into the plant genome, these results suggested that sequence information on those vectors, especially the ones within T-DNA regions, was responsible for the different expression patterns of LRP1::FT. Thus, we compared the sequences of pPZP212 and pCGN1547, especially those within the T-DNA regions.
Although pPZP212 and pCGN1547 contained similar configurations within their T-DNA regions, multiple cloning sites (MCS) and a positive selectable marker gene, nptII, the regulatory sequences of nptII were different. For the regulatory sequences, pCGN1547 used the promoter and terminator sequences of mannopine synthase (mas) (Fig. 2b), whereas pPZP212 used the 35S promoter and 3¢ untranslated region (UTR) sequences of the inclusion body matrix protein of 35S RNA (Fig. 2a). The 35S promoter is known to be a true enhancer, due to its enhancer sequence (nucleotides 417 to 86, relative to the transcription start) (Benfey et al. 1990a; Benfey et al. 1990b). The pPZP212 vector contains two copies of this 35S enhancer sequence (Fig. 2a), raising the possibility that the 35S enhancers in pPZP vectors trans activate an adjacent gene, as they did in the activation tagging screen (Weigel et al. 2000). It was previously shown that the 35S enhancer sequences upregulated a gene adjacent to the right border (RB), within up to 8.9 kb from a T-DNA integration site in the activation tagging screens (Weigel et al. 2000) (our unpublished data). Furthermore, the activation range could be larger than expected, as there has been a report that the 35S enhancers also activate a gene located 78 kb away from a T-DNA integration site in an activation tagging screen (Ren et al. 2003). These results strongly suggest that when the nptII gene in pPZP212 was introduced into the plant genome, the 35S enhancers upstream of the nptII activate an adjacent gene, possibly either a transgene in multiple cloning sites (MCS), or a
Fig. 2a–c Comparison of the T-DNA regions of pPZP212 (a), pCGN1547 (b) and SKI015 (c). pPZP212 and pCGN1547 used the same nptII gene, but used different regulatory sequences. The nptII gene in pPZP212 contained the sequence from Cauliflower mosaic virus 35S RNA, whereas the nptII gene in pCGN1547 contained the regulatory sequences of mannopine synthase (mas). The 35S promoter sequence used in pPZP212 contained two copies of the 35S enhancer, which was known to trans activate the transcription of an adjacent gene(s). The same 35S enhancer was used in SKI015, an activation tagging vector—except that SKI015 contained four copies of the 35S enhancer. MCS Multiple cloning sites
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gene(s) adjacent to a T-DNA insertion site in the genome (Fang et al. 1989; Ren et al. 2003; Weigel et al. 2000). It is probable that the same activation by the 35S enhancers is true for other plant transformation vectors carrying the 35S promoter, for example, the pCAMBIA series and pINDEX1. An important question is whether 35S enhancers in pPZP vectors behave similarly to those in SKI015, the activation tagging vector. In the SKI015, four copies of 35S enhancers are not associated with other promoter elements (Fig. 2c); thus, they may freely activate an adjacent gene(s). In contrast, the 35S enhancers in pPZP vectors are closely associated with a small fragment ( 84 to +43, relative to the transcription start site) of 35S promoter, which may prevent 35S enhancers from trans activating an adjacent gene. If the transcriptional enhancement of 35S enhancer is abolished due to this close association, expression of an adjacent gene is unaffected. However, it is likely that the 35S enhancers in pPZP vectors still affect, albeit weakly, the expression of an adjacent gene. Fang et al. (1989) showed that the 35S enhancer fragment used in their study, which is smaller than that of the SKI015, potentiated transcription when not associated with its 35S promoter element. Furthermore, a promoter activity of 35S promoter is largely dependent upon the upstream sequence including an enhancer, rather than the downstream sequence ( 84 to +43). A core sequence ( 46 to +8) in the downstream sequence has a weak promoter activity leading to lowlevel expression in roots (Benfey and Chua 1989); however, when the core sequence is fused with another promoter, the activity of the resulting chimeric promoter is similar to that of the promoter element fused with the core sequence. Thus, although the 35S enhancers in pPZP vectors are closely associated with the downstream sequence ( 84 to +43), the 35S enhancers likely has an enhancer activity leading to trans activation. Although we suggest the 35S enhancer is the main factor affecting the expression of a transgene, the combinatorial effects from the 35S minimal promoter, 35S UTR sequences, or distance between promoter elements in plant transformation vectors cannot be excluded. Further study on these elements will help identify the specific region causing the detrimental effects on the transgene expression. Affected expression mostly disappeared with modified vectors in which the 35S sequences were replaced To confirm that the 35S sequences used to regulate a selectable marker gene overrode the control of expression of the transgene, a set of modified vectors were constructed, where we replaced the 35S regulatory sequences. After removing the nptII gene, along with the 35S regulatory sequences, we introduced selectable marker gene cassettes containing the mas regulatory sequences used in pCGN1547 and named the modified
vectors pJHA212K, pJHA212G and pJHA212B, where K, G and B stand for kanamycin, gentamycin and BASTA resistance, respectively (Fig. 3a). Detailed procedures of the construction of the vectors are described in the supplementary material (Fig. S1). We tested the transformation efficiency of the modified vectors by introducing empty vectors into Arabidopsis. The transformation efficiencies of the empty pJHA212K, pJHA212B and pJHA212G were approximately 1.0– 2.5%, indicating that the replacement did not harm the T-DNA transfer of the modified vectors. The effect of substitution of the 35S sequences was tested by comparing the GUS staining pattern of UFO::GUS subcloned into pPZP222 and pJHA212B. UFO (Unusual Floral Organs) encodes an F-box-containing protein, which controls the meristem identity and organ primordia fate (Samach et al. 1999). Ethanolinduced GUS expression revealed that the expression of UFO in the central region of the shoot apical region (Deveaux et al. 2003). Thus, the same localization pattern, or at least a similar pattern, should be observed when a reporter assay is applied. If a reporter assay provides a very different result from its bona fide expression pattern, this may lead to misinterpretation of the role of the gene-of-interest. For a vector backbone containing the 35S regulatory sequences, we used the KB68 plasmid, where UFO::GUS was subcloned into pPZP222. To subclone the UFO::GUS gene into a vector not containing the 35S regulatory sequences, we isolated the UFO::GUS chimeric gene from the KB68 plasmid, which was subcloned into pJHA212B, resulting in pSYY003. We introduced KB68 and pSYY003 into Arabidopsis and compared their GUS staining patterns at the primary inflorescences of transgenic plants. The GUS staining patterns were arbitrarily classified into four classes; normal, weak ectopic, intermediate ectopic and strong ectopic patterns. Normal expression patterns reflected the expression pattern of the UFO obtained from the ethanol-inducible expression experiment (Deveaux et al. 2003), whereas ectopic patterns were designated ectopic GUS staining in floral organs and inflorescence, depending on the staining intensity. The presence of 35S regulatory sequences greatly affected the GUS staining patterns in KB68 plants (Fig. 3b). None of KB68 plants showed a normal pattern. Instead, 43 of 47 KB68 transgenic plants showed either strong or intermediate ectopic patterns of such strength that strong GUS staining was observed in whole inflorescences, including the pedicels (Fig. 3c). In contrast, pSYY003 plants showed similar results to those revealed by an ethanol-inducible GUS expression experiment on UFO (Deveaux et al. 2003; Laufs et al. 2003). Of 99 pSYY003 plants, 55 showed that GUS staining was specifically localized around the shoot apical region (Fig. 3c). Although some pSYY003 plants showed an ectopic UFO staining pattern, the staining intensity and percentage of ectopic expression were greatly reduced in pSYY003 plants, indicating that 35S
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Fig. 3 a Map of pJHA212 series vectors, the modified vectors based on pPZP212, where the regulatory sequences of the nptII gene of the pPZP212 vector were replaced with those from mannopine synthase. To provide multiple choices of selectable markers during plant transformation, aacC1 and BAR genes were also introduced into the modified vector, resulting in pJHA212G and pJHA212B. RB, right border; LB, left border b Distributions of GUS staining patterns in the primary inflorescences of KB68 and pSYY003 plants. Normal patterns showed staining in the shoot apical region. Weak ectopic patterns indicated additional staining in the petals of inflorescence, whereas intermediate ectopic patterns indicated additional staining in petals and carpels. Strong ectopic patterns denoted strong staining in all inflorescences. c GUS staining patterns of KB68 and pSYY003 plants. Although most pSYY003 plants showed either normal or weak ectopic patterns, the majority of KB68 plants exhibited either intermediate ectopic or strong ectopic patterns. S Shoot apical meristem. Bar 100 lm
2003). Additional sequences, or sequence elements other than that of the promoter, may be required to fully mimic endogenous UFO expression, as in the case of AG (AGAMOUS) (Busch et al. 1999). Nevertheless, although the UFO::GUS used in this study did not reflect the endogenous UFO expression, this experiment also showed the detrimental effect on the expression of transgenes by the 35S promoter, which was likely due to the 35S enhancer. Since we used the vectors of different configurations, another important question is whether the orientation of selectable marker gene cassettes is responsible for the altered expression. For example, the orientations of the selectable marker gene cassettes were converse in pJA1022 and pJA1102 constructs. If LRP1::FT was subcloned in the same direction while constructing these vectors, the 35S promoter was located adjacent to the LRP1 promoter in the pJA1022 vector, which may alter the expression pattern of LRP1::FT. The same is true for the KB68 and pSYY003 constructs. Thus, if the constructs used in this study were designed so that 35S promoter was located adjacent to the promoter of a transgene, it is possible to infer that the divergent repeat, not the 35S promoter, is the problem. However, the orientation of two promoters is not a likely factor affecting the interpretation of our results. This is because, although the direction of the selectable marker genes was converse, the promoters in a selectable marker gene and a transgene were not divergent, rather they were configured as a direct repeat or a convergent repeat (Fig. 4). For instance, pJA1102 and KB68 plants, in which a selectable marker gene and a transgene were configured as a direct repeat, still showed strong ectopic expression (Fig. 1). This suggested that the orientation of a selectable marker gene and a transgene is not responsible for the interference. sequences altered the GUS expression pattern in KB68 plants, as observed in pJA1022 plants. However, the UFO promoter used in this study was not sufficient to recapitulate the endogenous expression pattern of UFO, because the GUS staining pattern did not exactly match that observed from in situ hybridization (Laufs et al.
Conclusion Many researchers have used plant transformation vectors that carry the 35S promoter in the selectable marker
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Fig. 4 Comparison of the configurations of the sequence elements of the constructs used in this study and their correlation with altered expression in transgenic plants. The transgenes and selectable marker genes in pJA1022, pJA1102 and KB68 were arranged as direct repeats to avoid interference caused by a divergent configuration. Two promoters in the pSYY003 construct were convergent. In the cases of pJA1022 and KB68, the 35S promoters were located 1.3 and 2.4 kb downstream of the promoters of transgene, respectively; however, strong ectopic expression was still observed. Arrows denote the orientation of transcription. The LRP1 promoter, UFO promoter and GUS cDNA were 1.6, 3.8 and 1.8 kb long, respectively. The 35S enhancers in the 35S promoter were indicated as grey arrows
genes as this confers strong positive selection. We found that the 35S promoter trans affected and altered the expression pattern of transgenes and changed the phenotype of transgenic plants. However, when 35S sequences were replaced, the interference on the transgene by the 35S promoter mostly disappeared in transgenic plants. We suggest that this interference resulted from the 35S enhancer in the 35S promoter, as with the 35S enhancers in the activation tagging screen. Therefore, we suggest caution when selecting a plant transformation vector and in the interpretation of the transgenic results when using vectors carrying the 35S promoter within their T-DNA regions. Acknowledgements We are grateful to S.M. Hong for data collection. We thank J.H. Lee and J. Kim for their critical reading of the manuscript. S.Y. Yoo, S.K. Yoo and M.S. Choi were supported by the BK21 program. This work was supported by a grant from the Plant Signaling Network Research Center of the Korean Science and Engineering Foundation (to J.H.A.) and by a grant from Crop Functional Genomics Center (to J.H.A. and J.S.L.).
References Angenon G, Dillen W, Van Montagu M (1994) Antibiotic resistance markers for plant transformation. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp 1–13 Aukerman MJ, Amasino RM (1998) Floral induction and florigen. Cell 93:491–494 Benfey PN, Chua NH (1989) The CaMV 35S enhancer contains at least two domains wihch can confer different developmental and tissue-specific expression patterns. EMBO J 8:2195–2202
Benfey PN, Ren L, Chua NH (1990a) Combinatorial and synergistic properties of CaMV 35S enhancer subdomains. EMBO J 9:1685–1696 Benfey PN, Ren L, Chua NH (1990b) Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J 9:1677–1684 Busch MA, Bomblies K, Weigel D (1999) Activation of a floral homeotic gene in Arabidopsis. Science 285:585–587 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743 Colasanti J, Sundaresan V (2000) ‘Florigen’ enters the molecular age: long-distance signals that cause plants to flower. Trends Biochem Sci 25:236–240 Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469 Deveaux Y, Peaucelle A, Roberts GR, Coen E, Simon R, Mizukami Y, Traas J, Murray JA, Doonan JH, Laufs P (2003) The ethanol switch: a tool for tissue-specific gene induction during plant development. Plant J 36:918–930 Fang SC, Fernandez DE (2002) Effect of regulated overexpression of the MADS domain factor AGL15 on flower senescence and fruit maturation. Plant Physiol 130:78–89 Fang RX, Nagy F, Sivasubramaniam S, Chua NH (1989) Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. Plant Cell 1:141–150 Franck A, Guilley H, Jonard G, Richards K, Hirth L (1980) Nucleotide sequence of cauliflower mosaic virus DNA. Cell 21:285–294 Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 Jeong S, Trotochaud AE, Clark SE (1999) The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase. Plant Cell 11:1925–1934 Kang BH, Busse JS, Bednarek SY (2003) Members of the Arabidopsis dynamin-like gene family, ADL1, are essential for plant cytokinesis and polarized cell growth. Plant Cell 15:899– 913 Kardailsky I, Shukla VK, Ahn JH, Dagenais N, Christensen SK, Nguyen JT, Chory J, Harrison MJ, Weigel D (1999) Activation tagging of the floral inducer FT. Science 286:1962–1965 Laufs P, Coen E, Kronenberger J, Traas J, Doonan J (2003) Separable roles of UFO during floral development revealed by conditional restoration of gene function. Development 130:785–796 McBride KE, Summerfelt KR (1990) Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol 14:269–276 Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107:193–232
530 Ouwerkerk PB, de Kam RJ, Hoge JH, Meijer AH (2001) Glucocorticoid-inducible gene expression in rice. Planta 213:370–378 Ren S, Shippen DE, McKnight TD (2003) Activation of telomerase by synergistic action of a novel zinc finger protein and auxin. In: 14th International Conference on Arabidopsis Research. University of Wisconsin-Madison Samach A, Klenz JE, Kohalmi SE, Risseeuw E, Haughn GW, Crosby WL (1999) The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem. Plant J 20:433–445 Sessions A, Weigel D, Yanofsky MF (1999) The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia. Plant J 20:259–263 Smith DL, Fedoroff NV (1995) LRP1, a gene expressed in lateral and adventitious root primordia of arabidopsis. Plant Cell 7:735–745
Stam M, Mol JNM, Kooter JM (1997) The silence of genes in transgenic plants. Ann Bot 79:3–12 Takada S, Goto K (2003) Terminal flower2, an Arabidopsis homolog of heterochromatin protein1, counteracts the activation of flowering locus T by constans in the vascular tissues of leaves to regulate flowering time. Plant Cell 15:2856–2865 Weigel D, Ahn JH, Blazquez MA, Borevitz JO, Christensen SK, Fankhauser C, Ferrandiz C, Kardailsky I, Malancharuvil EJ, Neff MM, Nguyen JT, Sato S, Wang ZY, Xia Y, Dixon RA, Harrison MJ, Lamb CJ, Yanofsky MF, Chory J (2000) Activation tagging in Arabidopsis. Plant Physiol 122:1003– 1013 Zhao J, Peng P, Schmitz RJ, Decker AD, Tax FE, Li J (2002) Two putative BIN2 substrates are nuclear components of brassinosteroid signaling. Plant Physiol 130:1221–1229
