The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells

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1 The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells Shingo Nagaya, Kazue Kawamura, Atsuhiko Shinmyo and Ko Kato Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Corresponding author: , ; Fax, (Received October 1, 2009; Accepted December 15, 2009) Short Communication To express a foreign gene in plants effectively, a good expression system is required. Here we describe the identification of a transcriptional terminator that supports increased levels of expression. The terminators of several Arabidopsis genes were examined in transfected Arabidopsis T87 protoplasts. The heat shock protein 18.2 (HSP ) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mrna levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase ) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants. Keywords: Arabidopsis thaliana 3 End region Gene expression Heat shock protein gene Polyadenylation Terminator. Abbreviations : ADH, alcohol dehydrogenase ; CaMV, cauliflower mosaic virus ; CS, cleavage/polyadenylation site ; FUE, far upstream element ; Fluc, firefly luciferase ; GUS, β-glucuronidase ; HSP, heat shock protein ; MBP, maltose-binding protein ; NOS, nopaline synthase ; NUE, near upstream element ; OCS, octopine synthase ; RACE, rapid amplification of cdna ends ; rbcs, ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2b ; Rluc, Renilla luciferase ; UBQ5, ubiquitin 5 ; UTR, untranslated region. The ability to introduce foreign genes into plants provides a powerful tool for investigating the function of specific genes. Additionally, the generation of genetically modified plants may yield products with useful industrial or pharmaceutical applications. To develop good expression systems, efforts have been largely focused on the identification and characterization of highly expressed and/or regulated promoters. However, another important parameter of gene expression is the efficiency of terminator. The terminator regulates the level of expression by controlling transcriptional termination and 3 end processing of mrna. Different terminators strongly influence the level of gene expression ( Carswell and Alwine 1989, Ingelbrecht et al ). Most eukaryotic mrna is cleaved post-transcriptionally at a specific site between 10 and 30 nt downstream of a polyadenylation signal (a consensus AAUAAA sequence) in the 3 - untranslated region (3 -UTR) ( Proudfoot and Brownlee 1976 ). Subsequent to cleavage, a poly(a) tract with an average length of nt in mammals and nt in Saccharomyces cerevisiae, respectively, is added to the RNA at the cleavage site ( Brawerman 1981, Peltz and Jacobson 1993 ). This modification has been shown to affect its stability, capacity to be translated and nuclear to cytoplasmic export ( Zhao et al ). Here, we describe the identification of a transcriptional terminator that will permit more efficient transgene expression in plants. In contrast to the vast amount of work performed in yeast and animals, far less is known about mrna 3 end processing in plants. In silico analysis has shown that the polyadenylation signal is found in the predicted location in only 10% of 3 -UTRs in Arabidopsis thaliana ( Loke et al ). Mutagenesis of genes of numerous plant species and viruses revealed that plant terminators have three major elements: far upstream elements (FUEs), near upstream elements (NUEs; AAUAAA-like motifs) and a cleavage/polyadenylation site (CS). The NUE region is an A-rich element located within 30 nt of the poly(a) site ( Hunt 1994 ). The FUE region is a U- or UG-rich sequence that enhances processing efficiency at the CS ( Mogen et al. 1990, Rothnie 1996 ), which is itself a YA (CA or UA) dinucleotide within a U-rich region at which polyadenylation occurs ( Bassett 2007 ). In order to obtain terminators of various Arabidopsis genes, including the entire 3 -UTR and downstream sequence, we first identified their poly(a) sites. The genes analyzed included 1-aminocyclopropane-1-carboxylate synthase 2 (ACS2 ; At1g01480), alcohol dehydrogenase (ADH ; At1g77120), histone H4 (H4 ; At5g59690), heat shock protein 18.2 (HSP ; At5g59720), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2b (rbcs ; At5g38420) and ubiquitin 5 (UBQ5 ; At3g62250). Total RNA from Arabidopsis leaves or T87 suspension cells was subjected to 3 rapid amplification of cdna ends (RACE), then PCR products were cloned and poly(a) sites were mapped using at least 30 independent clones for each gene. Multiple poly(a) sites were identified in all genes (Supplementary Fig. S1A). For example, poly(a) sites from the HSP gene were identified at the following positions: 141 bp (two clones), 144 bp (one clone), Plant Cell Physiol. 51(2): (2010) doi: /pcp/pcp188, available online at The Author Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org 328

The HSP terminator enhances gene expression 155 bp (two clones), 158 bp (24 clones) and 162 bp (one clone), with numbering referring to bases downstream from the TGA or TAA stop codon, where the

S1B). In a similar manner, the ACS2, ADH, H4, rbcs and UBQ5 major poly(a) sites were mapped to nucleotide positions 329, 201, 182, 166 and 155, respectively.

2 The HSP terminator enhances gene expression 155 bp (two clones), 158 bp (24 clones) and 162 bp (one clone), with numbering referring to bases downstream from the TGA or TAA stop codon, where the first position of the stop codon (T in both cases) is designated as 3. Since most clones had a poly(a) site at nucleotide 158, we designated this as the major HSP poly(a) site (Supplementary Fig. S1B). In a similar manner, the ACS2, ADH, H4, rbcs and UBQ5 major poly(a) sites were mapped to nucleotide positions 329, 201, 182, 166 and 155, respectively. We then cloned 250 bp downstream of the stop codon of each gene (with the exception of the ACS2 gene, for which a 500 bp region was cloned). These fragments were used as terminators. To evaluate the influence of various terminators on transgene expression in plant cells, the terminator regions described above were fused to a β -glucuronidase (GUS ) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The nopaline synthase (NOS ) terminator from the Ti plasmid of Agrobacterium tumefaciens is widely used in plant expression vectors. Expression efficiency was assessed in transfected Arabidopsis T87 protoplasts ( Fig. 1 ). The ADH, HSP, H4 and UBQ5 terminators led to increased GUS expression relative to the NOS terminator. In particular, GUS activity with the HSP terminator was approximately 2.5-fold higher than with the NOS terminator. To confirm that the HSP terminator increases gene expression, we constructed plasmids with the HSP terminator fused to either Renilla luciferase (Rluc ) or maltose-binding protein (MBP ) reporter genes. Similar results (Supplementary Figs. S2A, B) were obtained as with the GUS reporter, suggesting that the ability of the HSP terminator to increase expression levels is not gene specific. In all expression experiments described thus far, transgenes were driven by the CaMV35S promoter. To determine whether or not the HSP terminator also functions in combination with other promoters, we fused the Arabidopsis elongation factor 1 α promoter (including the native first exon and intron) or the ubiquitin 1 promoter to the Rluc reporter gene, followed by the NOS terminator or the HSP terminator. We found that the HSP terminator was able to increase expression of the Rluc gene with either promoter (Supplementary Fig. S2C). These results suggest that up-regulation of expression by the HSP terminator is independent of the specific promoter and reporter gene used. The octopine synthase (OCS ) terminator from the Ti plasmid of A. tumefaciens and the CaMV35S terminator from CaMV are widely used in plant expression vectors. In particular, the CaMV35S terminator has been shown to be more effective than the NOS terminator in dicot (tobacco) and monocot (rice) plants ( Mitsuhara et al ). To investigate whether the HSP terminator enhances gene expression compared with the OCS and CaMV35S terminators, we carried out a transient expression assay using protoplasts prepared from dicot (Arabidopsis) and monocot (rice). In this comparison, the HSP terminator showed the highest RLUC activity in both dicots and monocots ( Fig. 2A ). Taken together, these results suggest that the HSP terminator is even more effective than previously established terminators Fig. 1 Efficiency of gene expression with various terminators in transfected Arabidopsis T87 protoplasts. The terminator regions were fused to a GUS gene under the control of the CaMV35S promoter. As an internal control for transient expression assays, the firefly luciferase ( Fluc ) gene was placed under the control of the CaMV35S promoter and the NOS terminator. Transfected protoplasts were incubated for 17 h at 25 C, and GUS and FLUC activity were then measured. Values relative to GUS/FLUC activity with the NOS terminator are shown. Mean values with the SD are shown for three individual transfected samples. Fig. 2 Efficiency of gene expression with the HSP terminator in transfected protoplasts. (A) RLUC/FLUC activity of Arabidopsis T87 (left) or rice (right) protoplasts transfected with CaMV35S-Rluc-NOS, CaMV35S-Rluc-OCS, CaMV35S-Rluc-35S T or CaMV35S-Rluc-HSP. CaMV35S-Fluc-NOS was used as an internal control in the transient expression assays. Values relative to RLUC/FLUC with the NOS terminator are shown. (B) Northern blot analysis of Rluc mrna levels in Arabidopsis T87 protoplasts transfected with CaMV35S-Rluc-NOS and CaMV35S-Rluc-HSP. Fractionated RNA (see Materials and Methods) was transferred to nylon membranes and hybridized with an Rluc probe. Rluc signal was calculated, with results shown as values relative to the Rluc of the NOS terminator. An ethidium bromidestained agarose gel showing rrna is shown as a loading control. 329

To investigate whether the HSP terminator affects accumulated mrna levels, total RNA harvested from Arabidopsis T87 protoplasts transfected with CaMV35S-Rluc-HSP reporter plasmid was subjected to

3 S. Nagaya et al. in contributing to increased expression levels in both dicot and monocot plants. Increased gene expression mediated by the HSP terminator could be caused by an increase in either mrna level or translational efficiency. To investigate whether the HSP terminator affects accumulated mrna levels, total RNA harvested from Arabidopsis T87 protoplasts transfected with CaMV35S-Rluc-HSP reporter plasmid was subjected to Northern blot analysis with an Rluc probe. The level of reporter mrna observed was 2.7-fold greater from HSP terminator than from NOS terminator constructs ( Fig. 2B ), indicating that the increased gene expression observed with the HSP terminator is caused by increased mrna accumulation. Previous work showed that a terminator sequence may have different effects depending upon the transient or stable nature of the transgene ( Ingelbrecht et al ). We therefore transformed A. thaliana plants using A. tumefaciens-mediated transformation, and two or four independent single copy transgenic Arabidopsis plants were identified for each vector (Supplementary Fig. S3). Transgene expression was determined by GUS mrna accumulation and GUS activity in leaf tissue of 4-week-old plants. Northern blot analysis showed that the HSP terminator was able to induce a higher level of GUS mrna expression ( Fig. 3A, lanes 3 6) compared with the NOS terminator ( Fig. 3A, lanes 1 and 2). We sought to optimize the system further by using a translational enhancer, the 5 -UTR of the tobacco ADH (NtADH) gene ( Nagaya et al ). This 5 -UTR has been shown to enhance GUS activity in transgenic tobacco plants ( Satoh et al ). The GUS activities of two independent single copy CaMV35S- GUS-NOS transgenic plants ( Nagaya et al ) were 21 and 28 nmol 4-methyl umbelliferone (4MU) min 1.(mg protein) 1 ( Fig. 3B, lines 17 and 24). The GUS activity of single copy CaMV35S-NtADH-GUS-NOS transgenic plants ( Fig. 3B, lines NOS 21 and 25) was approximately 20- to 30-fold higher than that of CaMV35S-GUS-NOS plants ( Fig. 3B, lines 17 and 24). CaMV35S-NtADH-GUS-HSP induced 2- to 3-fold higher GUS activity ( Fig. 3B, lines HSP 2, 30, 36 and 38) than did CaMV35S-NtADH-GUS-NOS (Fig. 3B, lines NOS 21 and 25). These data suggest that the HSP terminator is effective in contributing to increased expression of both transient and stable transgenes. Furthermore, CaMV35S-NtADH-GUS-HSP expression ( Fig. 3B, lines HSP 2, 30, 36 and 38) was approximately 60- to 100-fold higher than that of CaMV35S-GUS-NOS ( Fig. 3B, lines 17 and 24). When combined with the HSP terminator, a translational enhancer enhanced gene expression in an additive manner. Histochemical GUS analysis was also performed on transgenic plants containing the CaMV35S-NtADH-GUS gene followed by the NOS terminator or the HSP terminator. A similar staining pattern was observed with both constructs ( Fig. 4A ), the GUS expression pattern was not changed by either terminator. In addition to the constitutive CaMV35S promoter, we tested a further tissue-specific promoter, the NtADH promoter ( Nagaya et al ). We fused the NtADH promoter to the Fig. 3 GUS activity and mrna accumulation in single copy transgenic plants. (A) Northern blot analysis was performed using total RNA prepared from leaves of 4-week-old transgenic plants. Total RNA (5 µg) was fractionated on a 1.5% formaldehyde agarose gel. The fractionated RNA was transferred to nylon membranes and hybridized with the GUS probe. An ethidium bromide-stained agarose gel showing rrna is shown as a loading control. (B) Average GUS activity and standard deviation in 4-week-old transgenic plants. GUS activity is expressed in nanomoles of 4-methyl umbelliferone per minute per milligram of protein. Fig. 4 Histochemical GUS staining in 2-week-old transgenic plants containing the CaMV35S promoter or the NtADH promoter fused to the GUS reporter gene, followed by the NOS terminator or the HSP terminator. Scale bars indicate 1 mm. 330

4 The HSP terminator enhances gene expression GUS reporter gene, followed by the NOS terminator or the HSP terminator. GUS activity was observed in shoot apices and roots with the NOS terminator ( Fig. 4B ), consistent with previous results ( Dolferus et al ). This expression pattern did not change with the HSP terminator. We have identified a new terminator that can be used to increase gene expression in both monocot and dicot plants. The HSP terminator causes an increase in the level of accumulated mrna compared with transcripts with the NOS terminator ( Figs. 2B, 3A ). The reason for this difference is still unclear. It has been proposed that unstable primary transcripts are converted more quickly into stable mrnas in the presence of efficient cis -elements ( Carswell and Alwine 1989 ). Thus, one possibility is that the HSP terminator is more efficient than the NOS terminator at mrna 3 end formation, resulting in higher levels of accumulated mrna. Further studies are needed to determine whether the HSP terminator cis-elements affect the efficiency of cleavage and polyadenylation reactions. Materials and Methods Arabidopsis T87 and rice ( Oryza sativa ) suspension cells were grown as described previously ( Satoh et al ). Transgenic Arabidopsis plants were grown according to Nagaya et al. (2005). Transgenic Arabidopsis plants were generated on a wild-type ecotype Columbia (Col-0) background. The binary vector was introduced into A. tumefaciens strain LBA4404. Plant transformations were performed using the in planta transformation method ( Clough and Bent 1998 ). Protoplast isolation from Arabidopsis T87 and O. sativa suspension cells was performed as described previously ( Satoh et al ). Protoplasts were transfected using a polyethylene glycol protocol, as described previously ( Axelos et al ). The construction of the plasmids used in this study is described in the Supplementary data. The assay for GUS activity was performed as described previously ( Jefferson et al ). RLUC and FLUC activity was determined using the dual-luciferase reporter assay system (Promega, Madison, WI, USA). For Northern blot analysis of Rluc mrna levels in transfected Arabidopsis T87 protoplasts, protoplast cells were transfected with 20 µg of plasmid DNA. Transfected cells were collected after growing at 25 C for 17 h, and then a small sample was used to measure RLUC activity. The remaining protoplast was used for isolating total RNA, which was fractionated (10 µg) on a 1.5% formaldehyde agarose gel. Northern blot analysis was performed as described by Kodama et al. (2007). The intensities of Northern blot bands for the 1 kb Rluc mrna were quantitated by MultiGauge software version 3.1 (Fujifilm, Tokyo, Japan). Supplementary data Supplementary data are available at PCP online. Funding This work was supported by New Energy and Industrial Technology Development Organization [Green Biotechnology Program to K.K.]. Acknowledgments We thank Yoshiko Mano and Hiroko Katoh for poly(a) site mapping and GUS transient assays. We thank Nobuko Shizawa for histochemical GUS analysis. References Axelos, M., Curie, C., Mazzolini, L., Bardet, C. and Lescure, B. (1992 ) A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol. Biochem. 30 : Bassett, C.L. (2007 ) Regulation of Gene Expression in Plants: The Role of Transcript Structure and Processing. pp Springer Press, New York. Brawerman, G. (1981 ) The role of the poly(a) sequence in mammalian messenger RNA. Crit. Rev. Biochem. 10 : Carswell, S. and Alwine, J.C. (1989 ) Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol. Cell Biol. 9 : Clough, S.J. and Bent, A.F. (1998 ) Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. Plant J. 16 : Dolferus, R., Jacobs, M., Peacock, W.J. and Dennis, E.S. (1994 ) Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiol. 105 : Hunt, A.G. (1994 ) Messenger RNA 3 end formation in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45 : Ingelbrecht, I.L., Herman, L.M., Dekeyser, R.A., Van Montagu, M.C. and Depicker, A.G. (1989 ) Different 3 end regions strongly influence the level of gene expression in plant cells. Plant Cell 1 : Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987 ) GUS fusions: β -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6 : Kodama, Y., Nagaya, S., Shinmyo, A. and Kato, K. (2007 ) Mapping and characterization of DNase I hypersensitive sites in Arabidopsis chromatin. Plant Cell Physiol. 48 : Loke, J.C., Stahlberg, E.A., Strenski, D.G., Haas, B.J., Wood, P.C. and Li, Q.Q. (2005 ) Compilation of mrna polyadenylation signals in Arabidopsis revealed a new signal element and potential secondary structures. Plant Physiol. 138 : Mitsuhara, I., Ugaki, M., Hirochika, H., Ohshima, M., Murakami, T., Gotoh, Y., et al. (1996 ) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol. 37 : Mogen, B.D., MacDonald, M.H., Graybosch, R. and Hunt A.G. (1990 ) Upstream sequences other than AAUAAA are required for efficient messenger RNA 3 -end formation in plants. Plant Cell 2 : Nagaya, S., Kato, K., Ninomiya, Y., Horie, R., Sekine, M., Yoshida, K., et al. ( 2005 ) Expression of randomly integrated single complete copy transgenes does not vary in Arabidopsis thaliana. Plant Cell Physiol. 46 :

5 S. Nagaya et al. Nagaya, S., Nakai, Y., Kato, K., Sekine, M., Yoshida, K. and Shinmyo, A. (2000 ) Isolation of growth-phase-specific promoters from cultured tobacco cells. J. Biosci. Bioeng. 89 : Peltz, S.W. and Jacobson, A. (1993 ) mrna turnover in Saccharomyces cerevisiae. In Control of Messenger RNA Stability. Edited by Belasco, J. and Brawerman, G. pp Academic Press, New York. Proudfoot, N.J. and Brownlee, G.G. (1976 ) 3 Non-coding region sequences in eukaryotic messenger RNA. Nature 263 : Rothnie, H.M. (1996 ) Plant mrna 3 -end formation. Plant Mol. Biol. 32 : Satoh, J., Kato, K. and Shinmyo, A. (2004 ) The 5 -untranslated region of the tobacco alcohol dehydrogenase gene functions as an effective translational enhancer in plant. J. Biosci. Bioeng. 98 : 1 8. Zhao, J., Hyman, L. and Moore, C. (1999 ) Formation of mrna 3 ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mrna synthesis. Microbiol. Mol. Biol. Rev. 63 :

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