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First published online October 15, 2004; 10.1104/pp.104.051508 Plant Physiology 136:3550-3561 (2004) © 2004 American Society of Plant Biologists Untranslated Regions from C4 Amaranth AhRbcS1 mRNAs Confer Translational Enhancement and Preferential Bundle Sheath Cell Expression in Transgenic C4 Flaveria bidentis1Department of Biological Sciences, The State University of New York at Buffalo, Buffalo, New York 14120 (M.P., A.C.C., J.O.B.); Department of Biology, Capital Normal University, Beijing 100037, China (L-P.Y.); and Commonwealth Scientific and Industrial Research Organization Division of Plant Industry, Canberra 2601, Australia (S.A., W.C.T.)
Many aspects of photosynthetic gene expression are posttranscriptionally regulated in C4 plants. To determine if RbcS mRNA untranslated regions (UTRs) in themselves could confer any characteristic C4 expression patterns, 5'- and 3'-UTRs of AhRbcS1 mRNA from the C4 dicot amaranth were linked to a gusA reporter gene. These were constitutively transcribed from a cauliflower mosaic virus promoter and assayed for posttranscriptional expression patterns in transgenic lines of the C4 dicot Flaveria bidentis. Three characteristic C4 expression patterns were conferred by heterologous AhRbcS1 UTRs in transgenic F. bidentis. First, the AhRbcS1 UTRs conferred strong translational enhancement of gusA expression, relative to control constructs lacking these UTRs. Second, while the UTRs did not appear to confer tissue-specific expression when analyzed by  -glucuronidase activity assays, differences in gusA mRNA accumulation were observed in leaves, stems, and roots. Third, the AhRbcS1 UTRs conferred preferential gusA expression (enzyme activity and gusA mRNA accumulation) in leaf bundle sheath cells. AhRbcS1 UTR-mediated translational enhancement was also observed in transgenic C3 plants (tobacco [Nicotiana tabacum]) and in in vitro translation extracts. These mRNAs appear to be translated with different efficiencies in C4 versus C3 plants, indicating that processes determining overall translational efficiency may vary between these two categories of higher plants. Our findings suggest that the AhRbcS1 5'-UTR functions as a strong translational enhancer in leaves and other tissues, and may work synergistically with the 3'-UTR to modulate overall levels of Rubisco gene expression in different tissues and cell types of C4 plants.
Flaveria bidentis and amaranth (Amaranthus hypochondriacus) are both dicotyledonous plants that utilize the C4 pathway of photosynthesis. This specialized CO2 fixation pathway requires the abundant expression of genes encoding the various C4 enzymes in one of two leaf cell types. One set of CO2 fixation enzymes, including Rubisco, accumulates only in leaf bundle sheath (bs) cells, while others, such as phosphoenolpyruvate carboxylase, are produced only in leaf mesophyll (mp) cells. In all C4 species, the various photosynthetic enzymes are regulated independently (Furbank and Taylor, 1995  ; Berry et al., 1997; Sheen, 1999) yet share many characteristic gene expression patterns required for their function in CO2 fixation. These include very high levels of expression, localization to specific cell types within leaves or other photosynthetic tissues, and regulation by light. For many C4 genes, including those encoding the Rubisco small subunit (SSU; encoded by nuclear RbcS genes) and large subunit (encoded by plastid rbcL genes), posttranscriptional control processes have been implicated in one or more aspects of their regulation (Furbank and Taylor, 1995; Berry et al., 1997; Sheen, 1999).
In all plants, the Rubisco SSU is encoded by a multigene family (Dean et al., 1989
Many aspects of Rubisco gene expression are common to both C3 and C4 plants, the most notable of these being expression primarily in photosynthetic leaves and cotyledons, and regulation by light within these tissues (Gilmartin et al., 1990
The aspect of Rubisco regulation that is most characteristic of C4 plants, including F. bidentis and amaranth, is that in mature leaves and cotyledons genes encoding both Rubisco subunits are selectively down-regulated in mp cells, while continuing high levels of expression in bs cells. The end result is that Rubisco, an abundant enzyme in all photosynthetic cells of C3 plants, accumulates only in the bs cells of C4 plants, with little or no accumulation of Rubisco transcripts or subunit proteins in mp cells. In the C4 dicot amaranth, posttranscriptional regulation contributes substantially to the establishment and maintenance of bs-specific RbcS and rbcL gene expression during C4 leaf development (Boinski et al., 1993
To better understand the role of posttranscriptional regulation in determining highly abundant cell type and tissue-specific Rubisco gene expression in C4 plants, and the possible conservation of regulatory mechanisms among C4 dicots, we have prepared posttranscriptional gene expression constructs derived from the amaranth AhRbcS1 gene (Corey et al., 1999
Posttranscriptional Gene Expression Constructs Derived from AhRbcS1 mRNA
To investigate the roles of 5'- and 3'-UTRs in the posttranscriptional regulation of C4 RbcS gene expression, tagged RNA expression constructs were prepared from AhRbcS1 mRNA sequences (Corey et al., 1999
AhRbcS1-5'-UTR-gusA-3'-UTR Expression Levels in Leaves of Transgenic C4 Plants
For production of transgenic plants, the AhRbcS1 UTR-derived expression constructs and the control construct described above were inserted into the binary vector pGA482 (An, 1987 Overall levels of GUS activity were significantly higher in leaves of F. bidentis plants transformed with the CaMV/AhRbcS1-5'-UTR-gusA-3'-UTR expression construct (lines designated FbA53), relative to control plants (lines designated CaMV-GUS) expressing the gusA gene alone (lacking the AhRbcS UTRs) from the CaMV promoter. As shown in Figure 2A, GUS activity levels in leaves ranged from 18- to 250-fold higher in the FbA53 lines, relative to the control CaMV-GUS lines. Despite the great differences in gusA expression levels observed between the experimental and control plants, accumulation levels of gusA mRNA did not differ significantly between the two plant groups. Although there were some differences in mRNA accumulation between individual transgenic plants (Fig. 2B), when standardized to 18S rRNA levels the amount of gusA mRNA in the leaves of FbA53 and CaMV-GUS plants were approximately equal. These results clearly show that the enhanced levels of gusA expression in the FbA53 leaves, relative to the CaMV-GUS control leaves, was not due to increased levels of gusA mRNA accumulation.
When GUS activity levels in leaves of transgenic plants were standardized to levels of gusA mRNA accumulation and averaged for six FbA53 and three control CaMV-GUS lines (from lines shown in Fig. 2, A and B, and in Fig. 3), there was an average 48-fold enhancement of translation from the transcripts containing the AhRbcS1 UTRs, relative to control gusA transcripts lacking these UTRs (Fig. 2C). To alleviate concerns about potential background in reporter gene assays or hybridization analysis, wild-type (nontransformed) plants showed no discernable GUS activity in fluorescent 4-methyl umbelliferyl -D-glucuronide (MUG) assays or any hybridization to the gusA DNA fragment probe in slot-blot or northern analysis (wt in Fig. 2, A and B, and data not shown). It is clear that the construct containing the C4 AhRbcS1 UTRs was expressed more efficiently than the control construct lacking the UTRs in the transgenic C4 lines. Both expression cassettes were under control of the constitutive CaMV promoter, and enhanced expression occurred independently of gusA mRNA accumulation levels. Based on these findings, we conclude that high levels of expression from the AhRbcS1-5'-UTR-gusA-3'-UTR cassette were due to enhanced levels of translation in leaves of the transgenic F. bidentis plants.
Visual examination of various tissues isolated and stained for GUS activity from several transgenic plants indicated that, while overall levels of GUS activity were much higher in FbA53 than in control plants, both constructs expressed the gusA transgene throughout the plants. The quantitative data shown in Figure 3 are representative of these plants. Quantitative MUG activity assays revealed that enhanced levels of GUS activity from the CaMV/AhRbcS1-5'-UTR-gusA-3'-UTR expression cassette occurred in all tissues of the FbA53 plants examined, including photosynthetic leaves as well as nonphotosynthetic roots (Fig. 3A). It is especially interesting to note that, while GUS activity levels were enhanced to similar levels in the leaves, stems, and roots of FbA53 transgenic plants, gusA mRNA levels produced from the CaMV/AhRbcS1-5'-UTR-gusA-3'-UTR expression cassette were significantly lower in the nonleaf tissues of these plants (when standardized to levels of 18S rRNA; Fig. 3B). Relative to levels in leaves, gusA-containing transcripts were reduced greater than 6-fold in stems and greater than 20-fold in roots. In contrast, gusA mRNA levels in the CaMV-GUS control plants were present at similar levels in all three tissues. Taken together, it appears that some degree of tissue-specific regulation of gusA mRNA accumulation was conferred by the AhRbcS1 UTRs in the transgenic F. bidentis plants. Since the gusA gene was transcribed from a constitutive CaMV promoter, it is likely that the tissue-specific differences in gusA mRNA accumulation were regulated posttranscriptionally, through differential stabilization of the hybrid AhRbcS1 5'-UTR-gusA-3'-UTR mRNAs. In addition, it is clear that synthesis of GUS from these mRNAs in FbA53 transgenic plants was enhanced to similar high levels in leaves, stems, and roots, independent of levels of gusA transcripts that accumulated in these tissues. This suggests that rates of GUS protein synthesis and levels of mRNA accumulation are controlled by separate determinants in this transgenic C4 plant system. Visual observation of GUS-staining patterns within leaf cross-sections from transgenic FbA53 plants showed higher GUS activity in bs cells than in mp cells (Fig. 4A). In contrast, cross-sections from CaMV-GUS control plants showed homogenous (although much lower overall) levels of GUS staining in all leaf cell types (Fig. 4B). In situ hybridization using an antisense RNA probe of the gusA-coding region indicated patterns of gusA mRNA accumulation for the transgene mRNAs that were reflective of the GUS activity staining patterns. Higher levels of gusA mRNA accumulation were observed in leaf bs cell than in mp cells of the FbA53 lines (Fig. 4D), while similar amounts of gusA mRNA were present in both leaf cell types of CaMV-GUS lines (Fig. 4E). In control hybridizations with adjacent serial sections, FbRbcS1 mRNAs (detected using an FbRbcS1 antisense probe) were fully bs specific (Fig. 4F). Based on these findings, we conclude that gusA mRNAs containing the AhRbcS1-derived UTRs were preferentially expressed in bs cells, primarily at the level of mRNA accumulation, in leaves of the heterologous F. bidentis plants.
AhRbcS1 UTRs Confer Translational Enhancement in Other Plant Systems The AhRbcS1 UTR sequences conferred enhanced rates of translation in the transgenic F. bidentis plants, relative to plants lacking these UTRs. While this is not surprising, considering that these mRNAs encode photosynthetic proteins that accumulate to very high levels in plant cells, enhanced translational ability represents a very interesting and significant property of the C4 AhRbcS1 transcripts. To determine if the enhancement of gene expression conferred by the AhRbcS1 UTRs was specific to expression in the C4 dicot F. bidentis or if increased translation could occur in a C3 plant as well, binary vectors containing AhRbcS1 UTR-derived expression cassettes and the CaMV-GUS control cassette (Fig. 1) were transformed into the C3 dicot tobacco (Nicotiana tabacum). In addition to the pGG53 cassette, transgenic plants, stable lines transformed with expression constructs containing the AhRbcS1 5'-UTR alone (pGG5 cassette) or the 3'-UTR alone (pGG3 cassette), were also recovered. Transgenic lines of tobacco expressing all three AhRbcS1-derived cassettes and the control cassette were analyzed for GUS activity, gusA, and 18S rRNA levels, as described above. As in the transgenic F. bidentis plants, GUS activity from the CaMV/AhRbcS1-5'-UTR-gusA-3'-UTR construct, as well as the other constructs tested, was observed in all tissues of the plants, including leaves, stems, and roots. To quantify levels of translation from each construct, GUS activity in leaves was standardized to levels of gusA mRNA and averaged over at least five independent T0 lines transformed with each of the constructs. Figure 5 shows that there was an enhancement of GUS activity from a construct containing the AhRbcS1 5'- and 3'-UTRs (tobacco lines 5'3'GUSAh), and from a construct containing the 5'-UTR alone (5'GUSAh), relative to the control CaMV-GUS tobacco plants. However, this enhancement was not as strong as that observed in F. bidentis leaves (approximately 3-fold in transgenic tobacco lines expressing the pGG53 or pGG5 cassette).
Interestingly, the AhRbcS1 3'-UTR (tobacco line 3'-GUSAh) in itself appeared to repress expression in the C3 tobacco plants. When compared to expression in lines expressing the AhRbcS1-5'-UTR-containing constructs (5'GUSAh and 5'3'GUSAh) and even to the tobacco CaMV-GUS control lines, GUS activity relative to gusA mRNA accumulation in the 3'-UTR alone lines was considerably reduced. In the presence of the 5'-UTR, translation from the gusA mRNAs remained high. Such findings suggest that the AhRbcS1 3'-UTR, in the absence of the 5'-UTR, may act as an inhibitor of translation. The pGG53 and pBI221XS gusA expression cassettes shown in Figure 1 were inserted downstream of a T7 promoter to determine if AhRbcS1 UTR-mediated enhanced-translational ability could also occur in vivo in plant-derived cell-free translation extracts. Equal amounts of transcripts generated in vitro from each of these cassettes were translated in wheat germ cell-free translation extracts in the presence of 35S-Met. Relative rates of translation from each of these RNAs was determined by phosphorimager quantification of the 65-kD GUS protein product, following separation of the translation reactions by SDS-PAGE (Fig. 6A). To quantify the amount of GUS protein produced in each reaction relative to the amount of RNA present in the reactions more accurately, RNA was purified from aliquots of each extract following completion of the translation reactions. Relative amounts of gusA-containing RNA were then determined by northern-blot analysis, using a 32P-labeled gusA probe (Fig. 6B). Phosphorimager quantification demonstrated that approximately equal amounts of each transcript were present at the end of the reactions, ruling out the possibility that degradation of the transcripts could be a determining factor in the observed in vitro translation efficiencies.
Figure 6A shows that in vitro transcribed RNAs containing 5'- and 3'-UTRs derived from amaranth AhRbcS1 mRNAs showed enhanced levels of synthesis in vitro. Phosphorimager analysis revealed that there was a 7- to 8-fold increase in translation of the 68-kD GUS protein conferred by the AhRbcS1 UTRs (lane 3) compared to control RNA, which lacks RbcS UTRs (lane 2). In two independent experiments, when levels of GUS protein synthesized in vitro were standardized to amounts of RNA present, transcripts containing the AhRbcS1 UTRs were found to confer nearly identical increased levels of in vitro translation of the gusA coding region (7.52 and 7.48 in two experimental repeats), relative to control transcripts that lack the RbcS UTR (Fig. 6, bottom row). Taken together, findings from transgenic C4 and C3 plants, and the in vitro translation studies, demonstrate that UTRs derived from C4 RbcS mRNAs confer enhanced rates of translation to a gusA reporter gene. This enhancement can occur, with some variation in overall levels, in a wide range of experimental plant systems.
Although the C4 pathway of photosynthesis has been known for nearly four decades (Hatch and Slack, 1966 ; Hatch, 1997), underlying mechanisms responsible for determining the unique and complex gene expression patterns characteristic of this diverse group of plant species are not well understood. To date, evidence indicates that, during the evolution of C4 photosynthesis, multiple levels of regulation have been utilized and coordinated to achieve the cell-specific gene expression patterns required for the function of this specialized CO2 fixation pathway. Control of C4 gene expression at the level of transcription has been implicated in many studies (Nelson and Langdale, 1992; Furbank and Taylor, 1995; Berry et al., 1997; Sheen, 1999; Furbank et al., 2000; Matsuoka et al., 2001, Leegood, 2002; Shoichi and Burnell, 2003; Gowik et al., 2004). Photosynthetic promoters linked to reporter genes have reproduced some level of cell type-specific or tissue-specific expression patterns when these are expressed in C4 plants, although in some cases only partial bs or mp cell specificity has been obtained. Promoter regions from a maize (Zea mays) bs-specific RbcS gene, or mp-specific phosphoenolpyruvate carboxylase or pyruvate orthophosphate dikinase genes, conferred reporter gene expression that was highly specific to their respective cell types in transgenic maize plants (Matsuoka et al., 1993; Nomura et al., 2000a, 2000b). However, contribution from posttranscriptional control cannot be ruled out, since these expression constructs also contained 5'-UTR and coding regions of the mRNAs. Some sequences controlling bs cell specificity and high-level expression of the F. bidentis gene encoding the C4 NADP malic enzyme occur outside of the gene's promoter, within regions corresponding to the 5'- and 3'-UTRs and N-terminal coding regions of the Me1 transcript (Ali and Taylor, 2001a, 2001b). These findings suggest that regulation at transcriptional as well as posttranscriptional levels contribute to the C4 expression patterns of this bs-specific gene in F. bidentis. In amaranth, observations of AhRbcS gene expression during early leaf development (Ramsperger et al., 1996) and in photosynthetically inactive leaf regions (McCormac et al., 1997) indicate that control of bs cell specificity is initially regulated at the level of translation, followed by control at the level of transcription or transcript stability. While previous investigations have focused on the role of promoters and transcriptional control in the regulation of bs- or mp-specific gene expression, the potential involvement of regulatory sequences within the photosynthetic transcripts themselves have been largely overlooked. In this study, we examined how the UTRs from bs-specific AhRbcS1 mRNAs affect overall levels as well as tissue and cell type expression patterns in transgenic C4 plants and in other plant systems.
Findings presented here indicate that UTRs of the amaranth AhRbcS1 mRNAs conferred significantly enhanced levels of expression to a gusA reporter gene, relative to mRNAs that lacked these UTRs, in all tissues of transgenic F. bidentis plants. Significant but less dramatic enhancement was also observed in transgenic tobacco or in in vitro wheat germ extracts. Considering that RbcS mRNAs encode photosynthetic proteins that accumulate to very high levels in the chloroplasts of C4 as well as C3 plants (Edwards and Huber, 1981
Translational enhancement from AhRbcS1 mRNAs occurred very strongly in F. bidentis leaves and, to a lesser degree, in tobacco and in the cell-free wheat germ extracts. Differences in translational abilities for some transcripts are known to occur between different groups of plants; for example, transcripts containing the tobacco mosaic virus
At least some of the enhanced translational ability of the AhRbcS1-derived constructs may be due to the fact that their 5'-UTR has a better context for the initiator AUG than the CaMV-gusA control construct. Both constructs share identical gusA context downstream of the AUG codon (which is less optimal than Ah or Fb RbcS mRNAs; Corey et al., 1999
The 5'- and 3'-UTRs of AhRbcS1 mRNA in themselves were sufficient to confer tissue-specific accumulation patterns to gusA mRNA in transgenic F. bidentis plants. Highest levels of gusA transcripts in FbA53 plants occurred in leaves, with less accumulation in stems and significantly reduced accumulation in roots. This is reflective of relative levels of overall RbcS mRNA accumulation that occur in these tissue in intact amaranth plants (Nikolau and Klessig, 1987
Normally, specific degradation resulting in reduced levels of an mRNA would be expected to also result in reduced levels of the encoded protein. In the case of the FbA53 plants, reduced levels of gusA mRNA in stems and roots did not result in a corresponding lower levels of GUS activity in these tissues, suggesting there are separate determinants of GUS protein synthesis and mRNA accumulation in these transgenic C4 plants. The equal levels of GUS activity in these tissues did not result from saturation of the MUG assays, since increasing dilution of the extracts (up to 0.025-fold) produced the same relative levels of activity in the three tissues. Taking into consideration gusA transcript accumulation in the three tissues, together with the GUS activity assays, it appears that only a small amount of the more abundant gusA mRNAs in leaves of FbA53 plants were utilized for synthesis of the GUS protein, or else the less abundant transcripts accumulating in stems and roots were able to be utilized more efficiently. When the GUS activity data shown in Figure 3 were calculated relative to gusA mRNA levels, translation from AhRbcS1-5'-UTR-gusA-3'-UTR mRNA appeared to be at least 6- and 20-fold higher in stems and roots, respectively, relative to its translation in leaves. The reason for the disparity between mRNA accumulation and GUS activity in different tissues of FbA53 plants is not clear. Because of the highly enhanced protein synthesis ability of transcripts expressed from this construct, it is conceivable that levels of these hybrid mRNAs accumulating within the cells greatly exceeded the cells' ability to regulate their translation. This could result in the synthesis of equal amounts of the GUS enzyme from different levels of gusA transcript in all tissues of the FbA53 plants. An important consideration from these findings is that, while in the majority of situations measured levels of GUS enzymatic activity accurately reflect of levels of gusA mRNA accumulation and therefore gene expression (Jefferson, 1987 Preferential accumulation of the gusA mRNAs in bs cells was observed in cross-sections of mature leaves from FbA53 plants. GUS activity levels observed in these cross-sections were reflective of the amount of gusA transcript present in the two cell types, with higher levels present in bs cells than in other leaf cell types. Bs-preferential gusA mRNA accumulation or expression was not observed in leaf cross-sections from the CaMV-GUS control lines. Based on these analyses, we conclude that the AhRbcS1 UTRs were conserved enough and sufficient in themselves to confer at least partial C4-like bs cell-specific expression to the reporter gene in leaves of transgenic F. bidentis plants. Since the transgenes were constitutively transcribed, preferential gusA mRNA accumulation was regulated posttranscriptionally and is likely to be mediated by differential processing or stabilization of the AhRbcS1-derived transcripts in mature leaves of the FbA53 plants.
Previous studies have demonstrated that bs-specific accumulation of transcripts encoding both Rubisco subunits is posttranscriptionally regulated in mature leaves of C4 plants. In maize, nuclear run-on experiments have shown that RbcS mRNAs are transcribed in nuclei isolated from both bs and mp cells, whereas these transcripts accumulate only in bs cells of mature leaves (Schaffner and Sheen, 1991
The lack of full cell type-specific regulation for the CaMV/AhRbcS1-5'-UTR-gusA-3'-UTR construct in transgenic FbA53 leaves could be due to the heterologous UTR sequences present in the AhRbcS1 mRNA, which may not be not fully recognized by the endogenous C4 regulatory mechanisms of F. bidentis. In fact, initial evidence from the first regenerating leaves of new transgenic C4 lines indicates that constructs containing UTRs of the endogenous FbRbcS1 mRNAs show much tighter bs-specific expression than constructs derived from the heterologous AhRbcS1 mRNAs (M. Patel and J.O. Berry, unpublished data). Even small variations between these two plant species in terms of their mRNA regulatory sequences and the factors that recognize them might be expected to result in alterations to normal expression patterns, such as the greatly enhanced translation of the heterologous AhRbcS1-based constructs observed in the FbA53 plants. A comparison of UTR sequences from AhRbcS1 and two endogenous F. bidentis RbcS mRNAs (Corey et al., 1999
Findings presented here confirm that some aspects of RbcS gene expression in C4 plants are controlled posttranscriptionally and that regulatory processes are at least partially conserved between these two species of C4 dicot. Constitutively transcribed gusA mRNAs containing the AhRbcS1 UTRs showed enhanced overall levels of expression, tissue-specific mRNA accumulation, and preferential expression in leaf bs cells. The fact that full tissue- or bs cell-specific expression was not achieved from the AhRbcS1-derived construct in F. bidentis could be due to the heterologous origin of the UTR sequences. An alternative explanation is that additional mRNA sequences occurring elsewhere on full-length RbcS mRNAs might also be involved in mediating posttranscriptional photosynthetic gene expression in C4 plants. While cis-acting control regions often occur within the 5'- or 3'-UTRs of an mRNA, sequences within the coding region can also be involved (Gallie, 1993
Plant Materials and Growth Conditions All plants (transgenic and wild-type Flaveria bidentis, amaranth, and tobacco [Nicotiana tabacum]) were germinated and grown in growth chambers (14 h/d illumination at approximately 170–200 µmol photons m–2 s–1).
Total RNA was extracted from transgenic and wild-type plants, and northern- or slot-blot analysis was performed as previously described (Berry et al., 1985
All of the AhRbcS1-derived constructs used for this study were prepared from a modified form of the transient expression vector pBI221 (CLONTECH Laboratories, Palo Alto, CA). This vector, designated pBI221XS (Fig. 1), was produced by inserting a short DNA fragment containing an XhoI site into pBI221 at the SmaI site located just upstream of the nopaline synthase terminator. To prepare pGG3 (the AhRbcS1 3'-UTR construct), the full-length 3'-UTR was PCR amplified from an AhRbcS1 cDNA clone. The forward primer contained an XhoI site followed by an SstI site, followed by (from 5'–3') the first 20 bases of the 3'-UTR (from nt 600 through 619). The reverse primer contained (from 5'–3') the same two restriction cut sites, followed by the 20 bases immediately upstream of the polyA tail (antisense; from nt 728 through 709). The amplified 3'-UTR (3E103'XS) was then digested with XhoI, gel purified, and ligated into dephosphorylated pBI221XS. Electroporation of XL1-MRF cells (Stratagene) yielded a clone containing the 3'-UTR in correct orientation downstream of the gusA open reading frame (confirmed by restriction mapping and dideoxynucleotide sequencing), designated pGG3. To prepare pGG53 (the AhRbcS1 5'-UTR-gusA-3'-UTR construct) an oligonucleotide was prepared that contained a BamHI site at it's 5' end, followed by the 47 base AhRbcS1 5'-UTR sequence attached in frame to the first 21 bases of the sense strand of the gusA reporter gene-coding region. PCR was performed using this as the forward primer, together with a reverse primer specific for the 3' end of the AhRbcS1 3'-UTR, using the 3'-UTR-gusA construct pGG3 as the template. This amplified fragment was inserted into the BamHI/XhoI sites of pBI221XS from which the gusA gene had been removed. Following transformation, a clone containing the 5'- and 3'-UTRs in correct positions relative to the gusA open reading frame was identified by restriction mapping and dideoxynucleotide sequencing and designated pGG53 (Fig. 1). For pGG5, the 3'-UTR region was removed from the amplified pGG53 fragment described above before insertion into pBI221XS. Following transformation, a clone containing the 5'-UTR in correct position upstream of the gusA open reading frame was identified by restriction mapping and dideoxynucleotide sequencing and designated pGG5.
The AhRbcS1-derived expression cassettes contained within the constructs described above and shown in Figure 1 were all inserted into the binary vector pGA482 (An, 1987
For analysis of transgene expression, samples were harvested from young leaves of 1- to 3-month-old plants. To prepare protein extracts for fluorometric MUG assays, six leaf discs (6 mm diameter) were removed from each plant and placed into 500 µL of protein extraction buffer (50 mM Tris, pH 7.2, 1 mM EDTA, 10 mM Since the focus of these analyses was posttranscriptional control, GUS activity levels were standardized to levels of gusA mRNA for each sample. Since loading can vary somewhat between gel lanes or loading slots, gusA mRNA levels in each sample were first standardized to levels of 18S rRNA.
For localization of GUS enzyme activity, leaves (20–40 mm in length) of transgenic or wild-type F. bidentis were harvested from young (3–4-week-old) plants and stained with 5-bromo-4-chloro-3-indolyl
Sections from transgenic or wild-type F. bidentis leaves (20–40 mm in length) were prepared for in situ hybridization as described (Wang et al., 1992 All sections were visualized and images were captured using a Leica DM IRE HC inverted compound microscope equipped with a Retiga-cooled CCD camera (Leica Microsystems, Wetzlar, Germany).
The pGG53 and pBI221XS gusA expression cassettes were inserted into pBlueScript II SK+ for generation of RNA from the T7 promoter for use in in vitro translation assays. In vitro translation was performed in the presence of [35S]Met (PerkinElmer Life Sciences, Boston, MA) using wheat germ extracts (TNT system; Promega, Madison, WI). After the reactions were completed, aliquots were taken from each of the translation extracts for northern analysis of gusA transcripts present in the extracts. Translated proteins were run on 15% SDS-PAGE gel. RNAs were separated on an agarose-formaldehyde gel and analyzed by northern analysis using a gusA gene probe. Both gels were visualized and quantified using a phosphorimager and ImageQuant software. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY267350 and AY267351.
We are grateful to Jim Stamos for preparing the illustrations and Alan Siegel for capturing the microscope images. Sejal Patel and Xiowen Jiang provided excellent technical assistance. Received August 17, 2004; returned for revision September 7, 2004; accepted September 7, 2004.
1 This work was supported by the National Science Foundation (grant nos. INT 9724775 and MCB 0110411 to J.O.B.), the U.S. Department of Agriculture National Research Initiative (grant no. 2001–01825 to J.O.B.), and an academic exchange fellowship program from the University at Buffalo and Capitol Normal University (to L-P.Y.). 
2 Present address: Department of Cell and Structural Biology, University of Illinois, 190 Edward R. Madigan Laboratories (ERML), 1201 W. Gregory Drive, Urbana, IL 61801. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.051508. * Corresponding author; e-mail camjob{at}buffalo.edu; fax: 716–645–3369.
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enhancer are translated more efficiently in dicots than in monocots (Gallie et al., 1989
