The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells
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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, 630-0101 Japan
∗Corresponding author: E-mail, kou@bs.naist.jp; Fax, +81-743-72-5469
(Received October 1, 2009; Accepted December 15, 2009)
Short Communication
To express a foreign gene in plants effectively, a good a specific site between 10 and 30 nt downstream of a polyade-
expression system is required. Here we describe the nylation signal (a consensus AAUAAA sequence) in the 3′-
identification of a transcriptional terminator that supports untranslated region (3′-UTR) (Proudfoot and Brownlee 1976).
increased levels of expression. The terminators of several Subsequent to cleavage, a poly(A) tract with an average length
Arabidopsis genes were examined in transfected Arabidopsis of 200–300 nt in mammals and 70–90 nt in Saccharomyces
T87 protoplasts. The heat shock protein 18.2 (HSP) terminator cerevisiae, respectively, is added to the RNA at the cleavage site
was the most effective in supporting increased levels of (Brawerman 1981, Peltz and Jacobson 1993). This modification
expression. The HSP terminator increases mRNA levels has been shown to affect its stability, capacity to be translated
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of both transiently and stably expressed transgenes and nuclear to cytoplasmic export (Zhao et al. 1999).
approximately 2-fold more than the NOS (nopaline synthase) Here, we describe the identification of a transcriptional
terminator. When combined with the HSP terminator, terminator that will permit more efficient transgene expression
a translational enhancer increased gene expression levels in plants. In contrast to the vast amount of work performed
approximately 60- to 100-fold in transgenic plants. in yeast and animals, far less is known about mRNA 3′ end
processing in plants. In silico analysis has shown that the poly-
Keywords: Arabidopsis thaliana • 3′ End region • Gene adenylation signal is found in the predicted location in only
expression • Heat shock protein gene • Polyadenylation • ∼10% of 3′-UTRs in Arabidopsis thaliana (Loke et al. 2005).
Terminator. Mutagenesis of genes of numerous plant species and viruses
Abbreviations: ADH, alcohol dehydrogenase; CaMV, cauliflower revealed that plant terminators have three major elements:
mosaic virus; CS, cleavage/polyadenylation site; FUE, far far upstream elements (FUEs), near upstream elements (NUEs;
upstream element; Fluc, firefly luciferase; GUS, β-glucuronidase; AAUAAA-like motifs) and a cleavage/polyadenylation site (CS).
HSP, heat shock protein; MBP, maltose-binding protein; The NUE region is an A-rich element located within 30 nt of
NOS, nopaline synthase; NUE, near upstream element; OCS, the poly(A) site (Hunt 1994). The FUE region is a U- or UG-rich
octopine synthase; RACE, rapid amplification of cDNA ends; sequence that enhances processing efficiency at the CS (Mogen
rbcS, ribulose-1,5-bisphosphate carboxylase/oxygenase small et al. 1990, Rothnie 1996), which is itself a YA (CA or UA)
subunit 2b; Rluc, Renilla luciferase; UBQ5, ubiquitin 5; UTR, dinucleotide within a U-rich region at which polyadenylation
untranslated region. occurs (Bassett 2007).
In order to obtain terminators of various Arabidopsis
The ability to introduce foreign genes into plants provides a genes, including the entire 3′-UTR and downstream sequence,
powerful tool for investigating the function of specific genes. we first identified their poly(A) sites. The genes analyzed
Additionally, the generation of genetically modified plants may included 1-aminocyclopropane-1-carboxylate synthase 2 (ACS2;
yield products with useful industrial or pharmaceutical applica- At1g01480), alcohol dehydrogenase (ADH; At1g77120), histone
tions. To develop good expression systems, efforts have been H4 (H4; At5g59690), heat shock protein 18.2 (HSP; At5g59720),
largely focused on the identification and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit
highly expressed and/or regulated promoters. However, another 2b (rbcS; At5g38420) and ubiquitin 5 (UBQ5; At3g62250). Total
important parameter of gene expression is the efficiency of RNA from Arabidopsis leaves or T87 suspension cells was sub-
terminator. jected to 3′ rapid amplification of cDNA ends (RACE), then PCR
The terminator regulates the level of expression by control- products were cloned and poly(A) sites were mapped using at
ling transcriptional termination and 3′ end processing of mRNA. least 30 independent clones for each gene. Multiple poly(A)
Different terminators strongly influence the level of gene sites were identified in all genes (Supplementary Fig. S1A).
expression (Carswell and Alwine 1989, Ingelbrecht et al. 1989). For example, poly(A) sites from the HSP gene were identified at
Most eukaryotic mRNA is cleaved post-transcriptionally at the following positions: 141 bp (two clones), 144 bp (one clone),
Plant Cell Physiol. 51(2): 328–332 (2010) doi:10.1093/pcp/pcp188, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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328 Plant Cell Physiol. 51(2): 328–332 (2010) doi:10.1093/pcp/pcp188 © The Author 2009.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
Fig. 1 Efficiency of gene expression with various terminators in
as terminators. transfected Arabidopsis T87 protoplasts. The terminator regions were
To evaluate the influence of various terminators on trans- fused to a GUS gene under the control of the CaMV35S promoter.
gene expression in plant cells, the terminator regions described As an internal control for transient expression assays, the firefly
above were fused to a β-glucuronidase (GUS) gene under the luciferase (Fluc) gene was placed under the control of the CaMV35S
control of the cauliflower mosaic virus (CaMV) 35S promoter. promoter and the NOS terminator. Transfected protoplasts were
The nopaline synthase (NOS) terminator from the Ti plasmid of incubated for 17 h at 25°C, and GUS and FLUC activity were then
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Agrobacterium tumefaciens is widely used in plant expression measured. Values relative to GUS/FLUC activity with the NOS
vectors. Expression efficiency was assessed in transfected terminator are shown. Mean values with the SD are shown for three
Arabidopsis T87 protoplasts (Fig. 1). The ADH, HSP, H4 and individual transfected samples.
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 promot-
ers, we fused the Arabidopsis elongation factor 1α 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 Fig. 2 Efficiency of gene expression with the HSP terminator in
widely used in plant expression vectors. In particular, the transfected protoplasts. (A) RLUC/FLUC activity of Arabidopsis T87
CaMV35S terminator has been shown to be more effective than (left) or rice (right) protoplasts transfected with CaMV35S-Rluc-NOS,
the NOS terminator in dicot (tobacco) and monocot (rice) plants CaMV35S-Rluc-OCS, CaMV35S-Rluc-35S T or CaMV35S-Rluc-HSP.
(Mitsuhara et al. 1996). To investigate whether the HSP termina- CaMV35S-Fluc-NOS was used as an internal control in the transient
expression assays. Values relative to RLUC/FLUC with the NOS
tor enhances gene expression compared with the OCS and
terminator are shown. (B) Northern blot analysis of Rluc mRNA levels
CaMV35S terminators, we carried out a transient expression in Arabidopsis T87 protoplasts transfected with CaMV35S-Rluc-NOS
assay using protoplasts prepared from dicot (Arabidopsis) and and CaMV35S-Rluc-HSP. Fractionated RNA (see Materials and
monocot (rice). In this comparison, the HSP terminator showed Methods) was transferred to nylon membranes and hybridized with
the highest RLUC activity in both dicots and monocots (Fig. 2A). an Rluc probe. Rluc signal was calculated, with results shown as values
Taken together, these results suggest that the HSP terminator is relative to the Rluc of the NOS terminator. An ethidium bromide-
even more effective than previously established terminators stained agarose gel showing rRNA is shown as a loading control.
Plant Cell Physiol. 51(2): 328–332 (2010) doi:10.1093/pcp/pcp188 © The Author 2009. 329S. 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 transla-
tional 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 termina-
tor 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. 1989). We therefore
transformed A. thaliana plants using A. tumefaciens-mediated
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transformation, and two or four independent single copy
transgenic Arabidopsis plants were identified for each vector
(Supplementary Fig. S3). Transgene expression was deter-
mined 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). Fig. 3 GUS activity and mRNA accumulation in single copy transgenic
We sought to optimize the system further by using a transla- plants. (A) Northern blot analysis was performed using total RNA
tional enhancer, the 5′-UTR of the tobacco ADH (NtADH) gene prepared from leaves of 4-week-old transgenic plants. Total RNA (5 µg)
was fractionated on a 1.5% formaldehyde agarose gel. The fractionated
(Nagaya et al. 2000). This 5′-UTR has been shown to enhance
RNA was transferred to nylon membranes and hybridized with the
GUS activity in transgenic tobacco plants (Satoh et al. 2004).
GUS probe. An ethidium bromide-stained agarose gel showing rRNA is
The GUS activities of two independent single copy CaMV35S- shown as a loading control. (B) Average GUS activity and standard
GUS-NOS transgenic plants (Nagaya et al. 2005) were 21 and deviation in 4-week-old transgenic plants. GUS activity is expressed in
28 nmol 4-methyl umbelliferone (4MU) min−1.(mg protein)−1 nanomoles of 4-methyl umbelliferone per minute per milligram of
(Fig. 3B, lines 17 and 24). The GUS activity of single copy protein.
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 approxi-
mately 60- to 100-fold higher than that of CaMV35S-GUS-NOS
(Fig. 3B, lines 17 and 24). When combined with the HSP termi-
nator, 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
Fig. 4 Histochemical GUS staining in 2-week-old transgenic plants
pattern was observed with both constructs (Fig. 4A), the GUS containing the CaMV35S promoter or the NtADH promoter fused to
expression pattern was not changed by either terminator. the GUS reporter gene, followed by the NOS terminator or the HSP
In addition to the constitutive CaMV35S promoter, we tested terminator. Scale bars indicate 1 mm.
a further tissue-specific promoter, the NtADH promoter
(Nagaya et al. 2000). We fused the NtADH promoter to the
330 Plant Cell Physiol. 51(2): 328–332 (2010) doi:10.1093/pcp/pcp188 © The Author 2009.The HSP terminator enhances gene expression
GUS reporter gene, followed by the NOS terminator or the HSP
Funding
terminator. GUS activity was observed in shoot apices and roots
with the NOS terminator (Fig. 4B), consistent with previous This work was supported by New Energy and Industrial Tech-
results (Dolferus et al. 1994). This expression pattern did not nology Development Organization [Green Biotechnology Pro-
change with the HSP terminator. gram to K.K.].
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 accumu- Acknowledgments
lated mRNA compared with transcripts with the NOS termina- We thank Yoshiko Mano and Hiroko Katoh for poly(A) site
tor (Figs. 2B, 3A). The reason for this difference is still unclear. mapping and GUS transient assays. We thank Nobuko Shizawa
It has been proposed that unstable primary transcripts are for histochemical GUS analysis.
converted more quickly into stable mRNAs in the presence of
efficient cis-elements (Carswell and Alwine 1989). Thus, one
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