Untranslated Regions of a Mobile Transcript Mediate
RNA Metabolism1[C][OA]
Anjan K. Banerjee2, Tian Lin, and David J. Hannapel*
Plant Biology Major, Iowa State University, Ames, Iowa 50011–1100
BEL1-like transcription factors are ubiquitous in plants and interact with KNOTTED1 types to regulate numerous developmental processes. In potato (Solanum tuberosum subsp. andigena), the BEL1-like transcription factor StBEL5 and its Knox protein
partner regulate tuber formation by targeting genes that control growth. RNA detection methods and heterografting
experiments demonstrated that StBEL5 transcripts are present in phloem cells and move across a graft union to localize in
stolon tips, the site of tuber induction. This movement of RNA originates in leaf veins and petioles and is induced by a shortday photoperiod, regulated by the untranslated regions, and correlated with enhanced tuber production. Assays for RNA
mobility suggest that both 5# and 3# untranslated regions contribute to the preferential accumulation of the StBEL5 RNA but
that the 3# untranslated region may contribute more to transport from the leaf to the stem and into the stolons. Addition of the
StBEL5 untranslated regions to another BEL1-like mRNA resulted in its preferential transport to stolon tips and enhanced tuber
production. Transcript stability assays showed that the untranslated regions and a long-day photoperiod enhanced StBEL5
RNA stability in shoot tips. Upon fusion of the untranslated regions of StBEL5 to a b-glucuronidase marker, translation in
tobacco (Nicotiana tabacum) protoplasts was repressed by those constructs containing the 3# untranslated sequence. These
results demonstrate that the untranslated regions of the mRNA of StBEL5 are involved in mediating its long-distance transport,
in maintaining transcript stability, and in controlling translation.
As part of an elaborate long-distance communication system, plants have evolved a unique signaling
pathway that takes advantage of connections in the
vascular tissue, predominantly the phloem. This information superhighway has been implicated in regulating development, responding to biotic stress,
delivering nutrients, and as a vehicle commandeered
by viruses for spreading infections (Lough and Lucas,
2006). Numerous full-length transcripts have been
identified in the sieve elements of several plant species
(Asano et al., 2002; Vilaine et al., 2003; Omid et al.,
2007; Deeken et al., 2008; Gaupels et al., 2008), but only
six have been confirmed to be transported (Kehr and
Buhtz, 2008). Of these, three are from pumpkin
(Cucurbita maxima), CmGAIP, CmNACP, and CmPP16
(Ruiz-Medrano et al., 1999; Xoconostle-Cazares et al.,
1999; Haywood et al., 2005); DELLA-GAI is from
1
This work was supported by the U.S. Department of Agriculture
National Research Initiative Competitive Grants Program (grant no.
2008–02806) and the National Science Foundation Plant Genome
Research Program (grant no. 0820659).
2
Present address: Indian Institute of Science Education and
Research, Sutarwadi, Pashan, Pune 411021, India.
* Corresponding author; e-mail djh@iastate.edu.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
David J. Hannapel (djh@iastate.edu).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.144428
Arabidopsis (Arabidopsis thaliana; Haywood et al., 2005);
PFP-LeT6 is from tomato (Solanum lycopersicum; Kim
et al., 2001); and StBEL5, a BEL1-like transcription
factor, is expressed in potato (Solanum tuberosum
subsp. andigena; Banerjee et al., 2006a). Of this group,
information is available for StBEL5 RNA on the role of
the untranslated regions (UTRs) and photoperiod in
mediating movement and on the function of the mobile RNA (Banerjee et al., 2006a). Gel mobility-shift
assays have demonstrated the role of specific polypyrimidine tract-binding motifs in the phloem RNAs
of pumpkin, CmGAIP and CmPP16-1, that mediate
binding to RNA-binding proteins (Ham et al., 2009). In
this system, phloem RNAs and the CmRBP50 protein
provide the basis for a mobile ribonucleoprotein complex containing as many as 16 proteins.
In animals, mRNAs are transported within the cell
to facilitate their function. With the extensively studied
Staufen protein of Drosophila (St. Johnston et al., 1991;
Ferrandon et al., 1994), binding to the bicoid and oskar
mRNAs occurs to determine their localization in
the egg cell, either anterior or posterior. This directed localization involves RNA-protein, RNA-RNA
(Ferrandon et al., 1997), and protein-protein interactions (Elvira et al., 2006) during transport and repression of translation of the mRNAs. Repression of
translation ensures that the mobile mRNAs are functional only at the target site (King et al., 2005). Commonly, it is the UTRs that function via protein
interactions in facilitating movement of a transcript
(Jansen, 2001), in mediating RNA stability (Derrigo
et al., 2000; Lee and Jeong, 2006), or in regulating the
efficiency of translation (Gualerzi et al., 2003; Barreau
Plant PhysiologyÒ, December 2009, Vol. 151, pp. 1831–1843, www.plantphysiol.org Ó 2009 American Society of Plant Biologists
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Banerjee et al.
et al., 2006). Posttranscriptional regulation mediated
by protein/mRNA complexes occurs widely among
eukaryotes (Keene and Lager, 2005; Lee and Schedl,
2006; Sanchez-Diaz and Penalva, 2006; Keene, 2007;
Pullmann et al., 2007; Halbeisen et al., 2008). Without
protection, naked cellular RNAs quickly fall prey to
degradative processes (Shyu et al., 2008).
While there are several reports on full-length mobile
RNAs in plants (for review, see Kehr and Buhtz, 2008),
there is a scarcity of information on the RNA sequence
that mediates this process. These sequences form
recognition stem/loop structures, designated “zip
code elements.” These binding motifs have been identified in the RNAs of animals and function in recognizing RNA-binding proteins (for review, see Jansen,
2001). Whereas these zip codes may be located anywhere in the transcript, they are most predominant in
the 3# UTR (Macdonald and Kerr, 1997; Chartrand et al.,
1999; Saunders and Cohen, 1999; Corral-Debrinski
et al., 2000; Thio et al., 2000). Viroid RNA motifs
have also been identified that facilitated selective RNA
movement through plant cells (Qi et al., 2004; Zhong
et al., 2007, 2008). These short sequences most likely
mimic endogenous plant RNA motifs that are recognized by cellular factors for transport.
The mRNA of potato that is the focus of this study,
designated StBEL5, is a member of the BEL1-like family
of transcription factors. This family of transcription
factors is ubiquitous among plant species, and these
proteins interact with KNOTTED1 types to regulate
numerous developmental processes (Müller et al., 2001;
Chen et al., 2003; Smith and Hake, 2003; Kumar et al.,
2007; Pagnussat et al., 2007; Kanrar et al., 2008; Ragni
et al., 2008). In potato, the BEL1-like transcription factor,
StBEL5, and its Knox protein partner, POTH1 (for
potato homeobox 1), regulate tuber formation by mediating hormone levels in the stolon tip (Rosin et al.,
2003; Chen et al., 2004). This change in hormone levels
leads to the creation of a strong sink organ, the tuber, by
activating cell division and enlargement of specific cells
of the stolon tip (Xu et al., 1998; Hannapel et al., 2004).
Upon this activation, abundant amounts of Suc are
Figure 1. Constructs for the functional analysis of the 5# and 3# UTRs of StBEL5 (A and B) and tuber yields (C) for transgenic lines
expressing these constructs. Constructs were driven by either the CaMV 35S (A) or the leaf-abundant GAS (B) promoter in the
binary vector pBI101.2 for movement assays. Transformation, regeneration, and screening of transgenic potato lines were
performed as described by Banerjee et al. (2006b). The StBEL5 5# UTR and 3# UTRs are 146 and 505 nucleotides in length,
respectively. Transgenic lines of potato that overexpress the chimeric constructs of StBEL5 listed in A (driven by the CaMV 35S
promoter) were evaluated for their effect on tuberization (C). The rate of tuberization (number of long days [LD] to tuberize) was
determined by the first appearance of tubers from among 20 replicates (C). The yield (g fresh weight plant21) of tubers was scored
for four plants after 14 d of short-day (SD) conditions (8 h of light/16 h of dark). In vitro conditions were the same as described
previously (Chen et al., 2003). For the soil-grown experiment, plants from one independent transgenic line per construct were
grown in 10-cm pots under long days (16 h of light/8 h of dark) until they reached the 16-leaf stage and then transferred to shortday conditions. After 28 d under short days, four plants from a high-expressing independent line for each construct were
evaluated for tuber yield (g fresh weight plant21). Wild-type plants were used as controls. SE values are shown, and asterisks
indicate significant differences from the control as determined by t test at a confidence interval of 95%. ND, Not detected; NOST,
nopaline synthase terminator.
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Untranslated Regions Mediate RNA Metabolism
transported to the stolon tip for the synthesis of starch
in the newly formed tuber.
RNA detection methods and heterografting experiments demonstrated that StBEL5 transcripts are present in phloem cells and move across a graft union to
localize in stolon tips, the site of tuber induction
(Banerjee et al., 2006a; Yu et al., 2007). This movement
originates in leaf veins and petioles and is induced by
a short-day photoperiod, regulated by the UTRs, and
correlated with enhanced tuber production. Whereas
photoperiod mediates the movement of StBEL5 RNA,
activation of transcription of the StBEL5 gene in leaves
is regulated by either red or blue light, regardless of
photoperiod or light intensity (Chatterjee et al., 2007).
This study demonstrates that one or both of the UTRs
of the StBEL5 mRNA are involved in mediating its
long-distance transport, in maintaining transcript stability, and in controlling its translation.
RESULTS
RNA-specific tag and a gene-specific primer from the
cds or the 3# UTR of StBEL5 RNA. Movement was
assessed for the BEL5 constructs driven by both the
CaMV 35S and GAS promoters. In potato, the GAS
promoter is most active in the minor veins of leaves
and its activity is generally not detected in roots,
stolons, or tubers (Fig. 2; Banerjee et al., 2006a).
Predicted representative models for these UTR chimeric RNAs derived from Mfold analyses (Fig. 3A, +5#
UTR and +3# UTR) indicate that, whereas they both
maintained the finger-like structure composed of the
UTRs (circles), this latter structure is reduced in complexity in the truncated RNAs relative to the fulllength transcript of StBEL5 (compare the +5# UTR and
+3# UTR models with the full-length model [arrows],
Fig. 3A).
For the GAS promoter lines, most of the RNA will
arise from the leaf (Fig. 2). It is assumed, however, that
the CaMV 35S promoter is active equally in leaves,
stems, and stolons, so any increase of RNA levels in
the stolon tips would indicate enhanced RNA trans-
Effect of the UTRs of the StBEL5 RNA on Tuber
Phenotype and Transcript Mobility
Previous work has shown that both UTRs of StBEL5
together affected RNA mobility (Banerjee et al., 2006a).
To determine the relative contributions of these two
distinct regions to transcript mobility, constructs were
made with the BEL5 coding sequence (cds) with one or
the other UTR attached and driven by either the
cauliflower mosaic virus (CaMV) 35S (Fig. 1A) or the
leaf-abundant galactinol synthase (GAS; Fig. 1B) promoter. As expected, transgenic lines containing each of
the constructs driven by the CaMV 35S promoter
exhibited enhanced tuber production (Fig. 1C). Soilgrown transgenic lines under short-day conditions
produced greater tuber yields after 28 d. Tuber yields
were increased by as much as 6-fold in the 5# UTR
lines relative to control lines after 4 weeks. For in vitrogrown plants, the transgenic lines produced tubers
under noninductive (long-day) conditions and as
much as a 3-fold increase in tuber yields under
short-day conditions (Fig. 1C). In addition to the effect
on tuberization, these transgenic lines exhibited an
overall average increase in plant height of 16% relative
to the control lines. Other than these observations, the
transgenic lines resembled the wild-type phenotype.
To facilitate the quantification of transcript mobility
for numerous transgenic constructs, an assay for movement through whole plants was utilized in this study
instead of grafting. This former approach has been
developed as an appropriate protocol for accurate and
reproducible mobility assays in potato (Banerjee et al.,
2006a). To assess the effect on movement, the relative
amount of transgenic RNA present in leaf and stolon
tips after 12 d of short-day conditions was quantified
in the transgenic lines containing the constructs listed
in Figure 1, A and B. Reverse transcription (RT)-PCR
was performed on three replicates using a transgenic
Plant Physiol. Vol. 151, 2009
Figure 2. GUS activity driven by the leaf-abundant promoter (Ayre et al.,
2003) for GAS in transgenic potato plants detected by histochemical
analysis. With transgenic potato plants containing a GAS::GUS construct,
GUS activity was detected in leaves but not in roots or stolons. Activity is
generally not detected in tubers, but in a few rare cases, GAS::GUS
activity was detected in trace amounts on knobby structures of irregularly
formed tubers. A, Transgenic plant grown in vitro under long-day
conditions. B, Enlargement of leaf from the plant in A. C, Stolon tips
from a short-day plant. D, Newly formed tubers. E, Primary root tip. F,
Lateral root tips. C and D are from a soil-grown plant. E and F are from an
in vitro-grown long-day plant. These samples are representative of
numerous ones that were examined. Bars = 2.0 mm.
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Banerjee et al.
Figure 3. Effects of UTRs on movement of the StBEL5 mRNA. Representative Mfold models (Zuker, 2003) of two RNA constructs
assayed for movement and the full-length StBEL5 RNA (A; from left to right): 5# UTR + cds, cds + 3# UTR, and full-length RNA of
StBEL5. In nearly all Mfold models, the full-length mRNA has both UTRs in close proximity (arrows, full-length model), forming a
multifingered structure. The BEL5 UTRs for each model are highlighted by solid circles. Quantification of movement was
performed on transgenic lines with the constructs shown in Figure 1, A and B, and on StBEL5 constructs with or without UTRs (D,
FL and cds, respectively), all grown under short-day conditions (8 h of light/16 h of dark). Relative levels of RNA were quantified
from total RNA extracted from both new leaves (white bars) and 0.5-cm samples from the tip of the stolon (black bars) from three
separate plants for each construct. Homogenous RT-PCR products were quantified using ImageJ software (Abramoff et al., 2004)
and normalized using rRNA values. SE values of three clones from one independent transgenic line per construct are shown. A
movement factor is provided for mobility assays above each set of constructs (B–D). The movement factor is equal to the relative
stolon tip RNA quantity divided by the relative leaf RNA quantity. One set of constructs was driven by the GAS promoter of C.
melo (B), whereas the other two sets were driven by the CaMV 35S promoter (C and D). The GAS promoter is most active in the
minor veins of leaves (Ayre et al., 2003; Fig. 2). [See online article for color version of this figure.]
port. Using the 35S promoter, an assay with a movement factor of approximately 1.0 would indicate no
movement (Fig. 3D, cds construct). In one case, the
movement factor for a negative control using the GAS
promoter was 0.05 (Banerjee et al., 2006a). With the
GAS promoter, both constructs exhibited enhanced
mobility, but the +3# UTR transcripts were transported
more efficiently than the +5# UTR transcripts (Fig. 3B).
The difference in RNA accumulation in leaves relative
to stolon tips between transgenic lines with the 3# UTR
and the 5# UTR constructs was 4.8-fold (Fig. 3B, 1.58 O
0.33). In other words, with the 3# UTR, almost 5-fold
more BEL5 RNA was being transported from leaf veins
to stolon tips than with the 5# UTR. When considering
movement of these same chimeric transcripts expressed by the CaMV 35S promoter, however, mobility
1834
for both construct types was less than optimum. With
this promoter, the 5# UTR transcript exhibits a movement factor of 1.48, whereas the 3# UTR transcript
exhibits a movement factor of 0.96 (Fig. 3C). Compare
these data with the movement factors of 3.62 and 0.96
for the positive and negative mobility controls, respectively (Fig. 3D). Either only a very slight increase in
mobility (5# UTR construct) or none at all (3# UTR
construct) was observed with these test constructs.
Enhancing the Mobility of Another BEL1-Like RNA
To determine if StBEL5 UTRs are sufficient to mediate the transport of a mRNA from the leaf through
the phloem to the stolon tip, a chimeric construct
containing both StBEL5 UTRs fused to the cds of
Plant Physiol. Vol. 151, 2009
Untranslated Regions Mediate RNA Metabolism
another potato BEL1-like RNA was created (Fig. 4A,
BEL14 + BEL5 UTRs). The sequence of StBEL14 (accession no. AF406700) was selected because the RNA
for this BEL1-like gene of potato was detected only at
very low levels in stolons and tubers (Chen et al., 2003)
and was not detected in stem phloem cells using lasercapture microdissection (Yu et al., 2007). As a baseline
control, a construct consisting of the full-length
StBEL14 sequence without StBEL5 UTRs was evaluated for movement (Fig. 4A, FL-BEL14). As a positive
control, movement was assayed for a transgenic line
characterized previously (Banerjee et al., 2006a) expressing the full-length StBEL5 transcript (Fig. 4A, FLBEL5). To ensure that the source of most of these
transgenic RNAs was the leaf (similar to the StBEL5
promoter), the leaf-abundant GAS promoter was used
for all three constructs (Fig. 4A). The RT-PCR quantification procedure was the same as described for
Figure 3. As expected, the most efficient movement
of transgenic RNA was observed with the full-length
StBEL5 RNA, with a movement factor of 0.75 (Fig. 4B,
FL-BEL5). The difference in RNA accumulation in
leaves relative to stolon tips between transgenic lines
with BEL14 plus the StBEL5 UTRs or BEL14 without
these UTRs is 2.7-fold (Fig. 4B, 0.38 O 0.14). With the
UTRs, almost 3-fold more BEL14 transcript is being
transported to stolon tips than without UTRs. In
correlation with this enhanced movement, tuber yields
for the BEL14 + BEL5 UTR lines were 4-fold greater
than those lines with the FL-BEL14 sequence (Fig. 4C).
Figure 4. A, Three RNA constructs driven by the leaf-abundant GAS promoter were tested for their capacity to move from leaf
veins to stolon tips: FL-BEL14 (contains BEL14 cds plus both native BEL14 UTRs), BEL14 + both StBEL5 UTRs replacing the
StBEL14 UTRs, and full-length StBEL5. StBEL14 was chosen as a test RNA because it is not abundant in stems or stolons (Chen
et al., 2003). B, Relative RNA accumulation in new leaves and 0.5-cm samples from the tip of the stolon was quantified in
transgenic lines expressing these chimeric transcripts. Transgenic plants were grown under short days (8 h of light/16 h of dark)
for 12 d, and the RNA was extracted from new leaves (white bars) and from 0.5-cm stolon tips (black bars) from three separate
plants for each construct. C, At the same time, tuber yields were scored for BEL14 lines with and without UTR fusions.
Homogenous RT-PCR products were quantified using ImageJ software (Abramoff et al., 2004) and normalized using rRNA values.
SE values of three clones from one independent transgenic line per construct are shown. A movement factor is provided for
mobility assays above each set of constructs (B). The movement factor is equal to the relative stolon tip RNA quantity divided by
the relative leaf RNA quantity. D and E, Representative Mfold models of the two BEL14 RNA constructs assayed for movement.
Models for the RNA structure of BEL14 + the BEL5 UTRs exhibited the same multifingered structure (D, arrow) formed by the
UTRs (D, solid circles) of the full-length StBEL5 transcript (Fig. 3A, full-length model, solid circles). All predicted Mfold models
for StBEL14 exhibited a stable, conserved motif arising from its cds (D and E, dotted circles). NOST, Nopaline synthase
terminator. [See online article for color version of this figure.]
Plant Physiol. Vol. 151, 2009
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Banerjee et al.
Mfold analysis of the BEL14 constructs used in this
experiment showed that fusion of the BEL5 UTRs
resulted in a representative model structure that
exhibited the multifingered structure (Fig. 4D, arrow)
characteristic of the full-length StBEL5 RNA (Fig. 3A,
full-length) but absent in the model for FL-BEL14 (Fig.
4E). In addition, 100% of the StBEL14 RNA models
derived from the Mfold analysis exhibited a stable
stem/loop motif arising from the cds (Fig. 4, D and E,
cds motif).
Factors That Affect the Stability of StBEL5 Transcripts
To determine if the UTRs of StBEL5 have an effect on
transcript turnover, a stability assay was employed
using in vitro-harvested shoot tips from transgenic
lines expressing either the full-length StBEL5 transcript or the cds alone. After 7 d of incubation in vitro
under short days, shoot tips were harvested, total
RNA was extracted, and the StBEL5 RNA was assayed
for RNA stability. Quantitative RT-PCR was used for
measuring mRNA half-lives because it is more sensitive than northern-blot hybridization. As described
before, transgenic RNA- and gene-specific primers
were used in the RT-PCR. Not surprisingly, the presence of the StBEL5 UTRs enhanced the stability of its
mRNA. Whereas approximately 60% of the transcript
without any UTRs remained after 4 h (Fig. 5A), those
transcripts that included both UTRs were approximately 90% intact after 4 h and did not drop below
60% until after 12 h (Fig. 5B). The half-life of the fulllength transcript was approximately 14 h, whereas for
the cds alone it was extrapolated to be 5 h.
Because movement of the StBEL5 RNA is regulated
by photoperiod (Banerjee et al., 2006a), the effect of
photoperiod on transcript stability was assessed.
Wild-type plants of the photoperiod-responsive potato
were grown under either long- or short-day conditions. Quantitative RT-PCR was performed as described previously except that a pair of gene-specific
primers for wild-type StBEL5 was used. The stability
assays revealed that StBEL5 RNA was more stable
after long-day than after short-day conditions (Fig.
5C). From the onset of the experiment, after short-day
conditions the StBEL5 transcript exhibited a steady
decrease in RNA integrity. A half-life of approximately
1.7 h was observed for StBEL5 RNA from short-daygrown plants. After 10 h, the RNA remaining had
decreased to less than 20% (Fig. 5C). Conversely, the
StBEL5 RNA extracted from long-day-grown plants
remained relatively stable through 2 h (approximately
75% remaining) and exhibited a half-life of approximately 5.0 h (Fig. 5C).
The Effect of the UTRs of StBEL5 RNA on Translation
Figure 5. Effects of the UTRs (A and B) and photoperiod (C) on the
stability of StBEL5 RNA. Transcript stability time lines for three different
RNA molecules, transgenic cds of StBEL5 (A), transgenic full-length
StBEL5 (B), and native StBEL5 (C), were determined in potato plants.
One-centimeter shoot tips from in vitro-grown plants were harvested,
RNA was extracted, and quantitative RT-PCR with gene-specific
primers was performed as described previously (Banerjee et al.,
2006a). The percentage of RNA remaining for each sample is relative
to the amount of RNA present when the experiment was started (0 h).
Samples were harvested from plants grown under short-day (SD) conditions (A and B) or both long-day (LD) and short-day conditions (C). The
arrows indicate the approximate half-life point for each experimental
time line. The UTRs of StBEL5 (accession no. AF406697) include 146
nucleotides from the 5# end and 505 nucleotides from the 3# end. The
CaMV 35S promoter was used for the transgenic lines in A and B.
To assess the effect of the UTRs of StBEL5 mRNA on
translation, the cds of GUS with and without the UTRs
was cloned into pBI221 and assayed in a transient
translation system in tobacco (Nicotiana tabacum) pro-
toplasts. This in vitro procedure allows for the rapid
and efficient analysis of numerous constructs. Four
constructs were analyzed: GUS + the 3# UTR of
StBEL5, the 5# UTR of StBEL5 + GUS, GUS plus both
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Untranslated Regions Mediate RNA Metabolism
UTRs attached, and the GUS cds alone (Fig. 6A).
Translation of GUS was reduced in the construct
containing the 5# UTR of StBEL5 by 12% relative to
GUS alone (Fig. 6B). With the GUS + 3# UTR and the 5#
UTR + GUS + 3# UTR constructs, translation was
repressed by 32% and 50%, respectively (Fig. 6B). As a
comparison, using in vitro systems with marker genes,
regulatory sequence from the transcript, and coexpression of established RNA-binding proteins, levels
of translational repression for four mRNAs, GluR2,
Vg1, Ringo/SPY, and ASH1, were 30% to 40%, 40%
to 60%, 70% to 90%, and 80% to 90%, respectively
(Colegrove-Otero et al., 2005; Huang et al., 2006;
Padmanabhan and Richter, 2006; Deng et al., 2008).
Using RT-PCR, transcript levels for GUS in transfected
cells were verified to be essentially equivalent for all
four constructs except GUS plus both UTRs (data not
shown). These transcripts were approximately onethird more abundant than the GUS RNA without
UTRs. This was consistent with the stability assays
for full-length StBEL5 RNA (Fig. 5, A and B). In Figure
5, BEL5 RNA containing both UTRs was approximately 30% more stable than cds transcripts (no UTRs)
over the 1.0- to 4.0-h harvests.
DISCUSSION
The mRNA of StBEL5 is one of the few full-length
mRNAs in plants that have been confirmed to move a
long distance through the phloem from leaf veins to
stolon tips underground (Banerjee et al., 2006a; Kehr
and Buhtz, 2008). This movement has been consistently associated with enhanced tuber production
(Banerjee et al., 2006a). Our results here confirm that
the UTRs of the StBEL5 RNA are involved in mediating this transport. Whereas either UTR alone may
enhance mobility (Fig. 3B), our results suggest that
both UTRs together contribute to optimum mobility of
the StBEL5 RNA. The contributing effects of the respective UTRs are not clear, but there are numerous
examples demonstrating the importance of the 3#
UTRs in recognizing RNA-binding proteins that regulate metabolism and movement (Ferrandon et al.,
1994; Corral-Debrinski et al., 2000; Padmanabhan and
Richter, 2006; Irion and St. Johnston, 2007). A careful
analysis of the 3# UTR of StBEL5 reveals the presence
of three clusters of conserved motifs (Fig. 7). These
include five polypyrimidine tracts, six poly-U motifs,
and eight novel UAGUA motifs. It is conceivable
that these motifs function in interactions with RNAbinding proteins to regulate StBEL5 RNA metabolism.
For example, PTB proteins are RNA-binding proteins
that bind to clusters of polypyrimidine tracts (Auweter
and Allain, 2008).
Adding both UTRs to another BEL RNA of potato
made it more mobile, targeted it to stolon tips, and
enhanced tuber formation (Fig. 4). Although very little
is known about this delivery system in plants, the
temporal and spatial coordination of the transport of
functional mRNAs is a well-documented phenomenon in biology (Kloc et al., 2002; King et al., 2005). For
example, in complexes with other proteins, the Staufen
family of proteins plays a key role in transporting
specific RNAs to the anterior or posterior end of the
egg cell in Drosophila. In this system, delivery of the
mRNAs for oskar and bicoid to one end or the other
defines the primary axis of the developing embryo (St.
Johnston et al., 1991). In Saccharomyces cerevisiae, ASH1
Figure 6. The effects of the UTRs of StBEL5 on the
translation efficiency of GUS in tobacco protoplasts.
A, Four constructs driven by the 35S promoter in
pBI221 were analyzed: GUS + the 3# UTR of StBEL5,
the 5# UTR of StBEL5 + GUS, GUS plus both UTRs
attached, and the GUS cds alone. B, The LUC gene in
pBI221 under the control of the CaMV 35S promoter
was included as an internal control. A relative value
for GUS expression was obtained by dividing the
GUS activity by the specific LUC activity. Each
transfection was performed three times. Data are
means 6 SE. NOST, Nopaline synthase terminator.
The levels of GUS translation have been adjusted to
reflect differences in RNA accumulation for each of
the constructs.
Plant Physiol. Vol. 151, 2009
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Banerjee et al.
Figure 7. Enlarged computer-generated version (Zuker, 2003) of the 3# UTR of StBEL5 from nucleotide (nt) 2,215 to 2,674
(arrows). Three types of conserved motifs are highlighted: poly-U (solid line), a unique UAGUA motif (double-striped line), and
the polypyrimidine tract, CUUCU (dotted line). The enlarged figure (at right) is derived from the Mfold model of full-length
StBEL5 RNA (at left and Fig. 3A). Full-length StBEL5 RNA is 2,716 nucleotides in length. The 3# UTR begins at nucleotide 2,215
(designated by the lower arrow). It should be noted, however, that the existence of these conserved motifs does not validate their
function. [See online article for color version of this figure.]
mRNA is localized to the bud tip of daughter cells,
where it plays key roles in development (Olivier et al.,
2005). The localization of these transcripts depends on
interactions between cis-acting localization elements
in ASH1 that recognize the RNA-binding protein,
She2p. The ASH1 mRNA contains four elements that
are essential for the proper localization of this transcript (Chartrand et al., 1999). Three of these elements
(E1, E2A, and E2B) are located within the coding
region of the ASH1 mRNA, whereas the remaining
element (E3) includes the termination codon and is
located primarily within the ASH1 3# UTR. These
localization elements were predicted to form RNA
secondary structures containing stem loops (Gonzalez
et al., 1999) that facilitate binding to She2p and are
conserved in other bud-localized mRNAs. A cis-acting
1838
element in the cds of the Arabidopsis FLOWERING
LOCUS T (FT) transcript facilitated movement of the
FT mRNA in Nicotiana benthamiana. A fusion of the FT
RNA with RNA of Potato virus X enhanced spread and
systemic infection of the virus relative to the control
(Li et al., 2009). For the GAI RNA of Arabidopsis,
motifs in the cds and the 3# UTR display functional
roles in regulating RNA movement (Huang and Yu,
2009).
In the oocyte of Xenopus laevis, numerous RNAs are
localized to generate developmental polarity along the
animal or vegetal axis (for review, see King et al., 2005).
Fifteen RNAs have been identified that are localized to
the animal hemisphere and 20 that localize to distinct
domains within the cortex. Vg1 RNA is transported to
the vegetal cytoplasm, where localized expression of
Plant Physiol. Vol. 151, 2009
Untranslated Regions Mediate RNA Metabolism
the encoded protein is critical for embryonic polarity.
Interaction between specific repeated motifs within
the 3# UTR and RNA-binding proteins mediates the
localization of Vg1 RNA to the vegetal pole of Xenopus
oocytes (Lewis et al., 2004). The Vg1 localization pathway is directed by key protein factors, such as the
RNA-binding proteins Vg1RBP/vera and PTB/
hnRNP I, which recognize and bind to these localization elements (Lewis et al., 2008). As many as 16
phloem proteins have been identified that are associated with mobile RNAs in pumpkin (Ham et al., 2009).
This ribonucleoprotein complex moves through a graft
union between pumpkin and cucumber (Cucumis
sativus), but the precise roles of the proteins in this
complex in transport and RNA metabolism are still not
clear.
Using an RNA stability assay, our results indicate
that the stability of StBEL5 RNA is photoperiod dependent (Fig. 5C) and mediated by its UTRs. That
UTRs enhanced RNA integrity is not an unusual
observation. UTRs are known to mediate transcript
degradation rates of numerous RNAs. A genome-wide
analysis of mRNA decay rates in Arabidopsis showed
that sequence elements in the 3# UTRs were enriched
in short- and long-lived transcripts (Narsai et al.,
2007). Light regulation of RNA stability has also
been documented. Transcripts of ferredoxin are stabilized in light and destabilized in dark by their association with polyribosomes and their accessibility to
ribonucleases. The element responsible for this light
regulation is the internal light-regulatory element located within the 5# UTR of the ferredoxin mRNA
(Hansen et al., 2001; Bhat et al., 2004). The stability of
the mRNA of the fatty acid desaturase, FAD7, is
reduced in response to light, whereas the transcript
is relatively stable under dark conditions (Collados
et al., 2006). Stability of the mRNA of one of the
Arabidopsis oscillator genes for the circadian clock,
CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), is stable in the dark and in far-red light but has a shorter
half-life in red and blue light (Yakir et al., 2007).
Light-mediated stability of CCA1 mRNA is an important mechanism for ensuring that the circadian
oscillator is accurately entrained by environmental
changes. StBEL5 mRNA accumulates in leaf veins
and petioles under the long days of summer, along
with sugars from photosynthesis, and is induced to
move during the onset of short days. In this system, it
is logical that signal mRNA accumulating under long
days has some degree of enhanced protection from
degradation until transport is activated by short days
late in the summer.
As previously discussed, the Staufen protein of
Drosophila binds to bicoid and oskar mRNAs to determine their localization in the egg cell and to repress
translation until this occurs (St. Johnston et al., 1991;
Ferrandon et al., 1994). Results from the translation
assay indicate that the 3# UTR of StBEL5 is involved in
the repression of translation (Fig. 6B). Whereas there
are numerous examples of the 3# UTR playing a role in
Plant Physiol. Vol. 151, 2009
the repression of translation of animal mRNAs (Gavis
and Lehmann, 1994; Kim-Ha et al., 1995; Huang et al.,
2006; Padmanabhan and Richter, 2006), there is very
little information on such a role for 3# UTRs in plants.
In contrast, the role of the 5# UTR in enhancing
translation has been well established (Dansako et al.,
2003; Patel et al., 2004; Dugré -Brisson et al., 2005;
Sugio et al., 2008). Because of its prominent role in
regulating tuber development and its capacity for
long-distance transport to stolon tips (Chen et al.,
2003; Banerjee et al., 2006a), control of translation of
the mRNA encoding the BEL5 transcription factor in
the correct place and at the correct time in potato is
imperative. Normally, the BEL5 promoter is very
active in leaf vasculature in response to light but is
inactive in stems (Chatterjee et al., 2007). As an example of what happens when misexpression occurs in
nontarget organs, consider the phenotype exhibited by
transgenic lines that constitutively overexpress the cds
(no UTRs) of StBEL5. Extreme phenotypes of these
lines are characterized by the formation of aerial
tubers from stems and by a reduction in the yield of
underground tubers (Fig. 8). Such extreme phenotypes
are not normally observed in transformants expressing the full-length StBEL5 construct (Banerjee et al.,
2006a).
Remarkably, translation repression occurs for all
four mobile nonplant mRNAs previously discussed,
oskar and bicoid mediated by Staufen, ASH1 mediated
by Kdh1p and Puf6p (Gu et al., 2004; Paquin et al.,
2007), and Vg1 mediated by ELAV (for embryonic
lethal and abnormal vision) proteins (Colegrove-Otero
et al., 2005). Vg1 RNA is abundant during the earliest
stages of oogenesis in Xenopus (Melton, 1987) but is not
Figure 8. The formation of aerial tubers (black arrows) from stem
axillary buds of a transgenic line of potato that constitutively overexpresses the cds (no UTRs) of StBEL5. Expression of the transgene was
driven by the CaMV 35S promoter. This BEL5 transgenic line, designated no. 20, exhibited an extreme phenotype characterized by the
formation of aerial stem tubers, stunted growth, and a reduction in the
yield of underground tubers (Chen et al., 2003). A leaf has been excised
(white arrow) to enhance visibility.
1839
Banerjee et al.
translated until the later stages of development during
localization to the vegetal cortex (Wilhelm et al., 2000).
In the cases of oskar and ASH1, repression is spatially
regulated (Micklem et al., 2000; Paquin and Chartrand,
2008). As has been postulated for StBEL5 RNA, repression of translation ensures that the mobile mRNAs
mentioned above, most of which encode developmentally important transcription factors, are functional
only at their target sites. Consistent with the aberrant
phenotype for the BEL5 overexpression line shown in
Figure 8, misexpression of Vg1 in the animal hemisphere results in severe patterning defects (Dale et al.,
1993).
The long-distance transport of StBEL5 RNA from
leaves to stolon tips to activate tuber formation in
potato represents a complex and dynamic signaling
system. In this model system of plants for non-cellautonomous movement of full-length mRNAs, it is
readily apparent that escort factors that facilitate
transport are not the only interactions putatively
mediated by UTRs that affect RNA metabolism and
target efficient expression of the mobile RNA. That
so many proteins have been identified in the ribonucleoprotein complex of a phloem-mobile RNA of
pumpkin (Ham et al., 2009) is consistent with this
multifaceted role of RNA-binding proteins in RNA
metabolism.
MATERIALS AND METHODS
Construct Design
For the transgenic lines in Figure 1A, both constructs, cds + 5 UTR and cds +
3 UTR, were PCR cloned downstream from the CaMV 35S promoter into the
XbaI/SacI site of the binary vector pCB201 (Xiang et al., 1999). For the GAS
promoter lines in Figure 1B, both UTR constructs were cloned into the XmaI/
SacI site downstream from the GAS promoter that had been cloned previously
into pBI101.2 (Banerjee et al., 2006a). For the StBEL14 constructs in Figure 4A,
full-length StBEL14 sequence (1,690 nucleotides including its native UTRs
[GenBank accession no. AF406700.1]; designated FL-StBEL14) was cloned into
the XbaI/SmaI site of pBI101.2 vector with the GAS promoter inserted
previously. For the BEL14 plus both BEL5 UTRs construct, cloning was
performed sequentially. First, the cds of a PCR-generated fragment of StBEL14
with the KpnI/BamHI site and the 500-nucleotide 3# UTR of StBEL5 with
BamHI and SacI sites were subcloned into pBluescript SK+. The KpnI/SacI
fragment of the BEL14 cds + the 3# BEL5 UTR fusion and a XmaI/KpnI
fragment of the 5# UTR of BEL5 were purified via restriction digestion and
then cloned into the XmaI/SacI site of pBI101.2 containing the GAS promoter
in a one-step ligation. Cloning of the full-length and cds constructs of StBEL5
driven by the CaMV 35S and GAS promoters, shown in Figures 3D and 4A,
respectively, was described previously (Banerjee et al., 2006a, 2006b).
For translation studies in the tobacco (Nicotiana tabacum) protoplasts, GUS
constructs were PCR cloned into the transient expression vector pBI221
(Clontech). First, the full-length StBEL5 3# UTR was cloned into the SacI site of
pBI221 to create the GUS + 3# UTR construct. The SacI primers used were
5#-AGTCCTGAGCTCGAAAGTCTCGTATTGATAGC-3# and 5#-GTCACTGAGCTCCTAATCTAATAATGATAGCAC-3#. The full-length 5# UTR of
StBEL5 was then cloned into the XbaI/BamHI site of the GUS + 3# UTR
construct to create the GUS + both UTRs construct. The primers used for this
cloning step were 5#-CTACTCTAGAGATAGTACTTATCTGCAA-3# with XbaI
at the 5# end and 5#-CATCGAGGATCCTTCTTTCTTCTTCGGATTTCTCCTTTTATCT-3# with BamHI at the 3# end. Finally, the 5# UTR + GUS construct
was created by cutting the full-length (both UTRs) vector with SacI to release
only the BEL5 3# UTR. All constructs were confirmed via sequencing at the
DNA Facility at Iowa State University.
1840
Plant Material and Generation of Transgenic Lines
Transformation was implemented on the photoperiod-responsive potato
(Solanum tuberosum subsp. andigena; Banerjee et al., 2006b). In photoperiodadapted genotypes like andigena, short-day photoperiods (less than 12 h of
light) are required for tuber formation, whereas under long-day conditions, no
tubers are produced. The in vitro transgenic potato plants were maintained in
a growth chamber (Percival Scientific) at 27°C with a photoperiod of 16 h of
light/8 h of dark and a fluence rate of 40 mmol m22 s21. Soil-grown plants were
maintained at 22°C in a growth chamber under either a long-day (16 h of
light/8 h of dark) or short-day (8 h of light/16 h of dark) photoperiod with a
fluence rate of 100 mmol m22 s21. Expression of the GUS reporter gene driven
by the GAS promoter (Fig. 2) was analyzed by incubating the samples
overnight at 37°C in GUS buffer containing 1.0 M NaHPO4, pH 7.0, 0.25 M
EDTA, pH 8.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide,
10% Triton X-100, 1 mg mL21 5-bromo-4-chloro-3-indolyl-b-D-GlcUA. Samples
were cleared with 100% ethanol and photographed employing a Nikon
COOLPXX995 digital camera.
Movement Assays
Transgenic lines expressing the various transcript constructs were driven
by either the CaMV 35S promoter or the leaf-abundant GAS promoter from
Cucumis melo (Ayre et al., 2003). Transgenic plants were grown under short or
long days for 12 d, and the RNA was extracted from 0.5-cm stolon tips and
new leaves from three separate plants for each construct. One-step RT-PCR
was performed using 20 ng of total RNA, a non-plant-sequence primer fused
to all transgenic RNAs, and a gene-specific primer from the test mRNA. Use of
the non-plant-sequence primer, NT-2, corresponding to sequence from the
NOS terminator (Banerjee et al., 2006a), makes it possible to discriminate
transgenic RNA from native BEL5 RNAs. The internal control for PCR was18S
rRNA. All PCRs were standardized and optimized to yield product in the
linear range, and conditions were adjusted based on the primers used. Genespecific primers for the BEL5 cds + 3# UTR and 5# UTR + BEL5 cds constructs
were 5#-TTTCTTTTGGGTTGGCTTGGAGTA-3# and 5#-GGGAGATTTTGGAAGGTTTG-3#, respectively. The conditions for RT-PCR were set as follows:
50°C for 30 min, 94°C for 2 min, 94°C for 15 s, 56°C for 30 s, 68°C for 30 s,
followed by one cycle of incubation at 68°C for 5 min. After determining the
linear range for all of the PCR products, 38 and 18 cycles were used for BEL5
and 18S rRNA amplifications, respectively.
Primers for the 18S rRNA were provided in the manufacturer’s kit
(Ambion Quantum RNA Universal 18S, catalog no. 1718). For constructs
driven by the GAS promoter, the PCR cycle numbers were adjusted to 40 for
BEL5 and to 17 for the 18S rRNA to be in the linear range. For analysis of the
full-length StBEL5 RNA, the conditions and primers were described previously (Banerjee et al., 2006a). For the GAS:StBEL14 quantitative RT-PCR
analysis, the StBEL14 gene-specific primer, 5#-CATCAATGGGGTTAACTACA-3#, and NT-2 were used. For quantitative analysis of BEL14 + UTRs
(Fig. 4B), the StBEL5 3# UTR primer, 5#-TTTCTTTTGGGTTGGCTTGGAGTA-3#,
and the NT-2 primer were used. The same RT-PCR conditions were used as
described previously, with the cycles adjusted to 32 for gene-specific RNA and
17 for the 18S rRNA. Homogenous PCR products were quantified using
ImageJ software (Abramoff et al., 2004) and normalized with the rRNA values.
SE values of three clones from one independent transgenic line per construct
are shown. A movement factor is provided for mobility assays in Figures 3
and 4. The movement factor is equal to the relative stolon tip RNA quantity
divided by the relative leaf RNA quantity. Structural modeling of mRNAs was
performed using Mfold software (Zuker, 2003). Predicted models were
evaluated for relative stability and frequency of conserved motifs. Based on
these criteria, representative models were presented for each mRNA sequence
(Figs. 3A and 4, D and E). Mfold models are based on statistical analysis and
do not represent the actual native structure of folded RNAs.
Stability Assays
Shoot tips of potato, approximately 2.0 cm in length, were excised and
harvested from aseptically grown 4-week-old wild-type or transgenic lines.
Excised shoot tips were then cultured in semisolid (0.2% Gelrite) growth
medium supplemented with Murashige and Skoog (1962) basal salt mixtures and 2.0% Suc. About 150 shoot tips were cultured for each experimental design with two replicates each. Shoot tips were cultured at 22°C
under either a long-day (16 h of light/8 h of dark) or a short-day (8 h of
Plant Physiol. Vol. 151, 2009
Untranslated Regions Mediate RNA Metabolism
light/16 h of dark) photoperiod. After 7 d in culture, 1.0-cm shoot tips were
excised and transferred to a 250-mL flask containing 80 mL of incubation
buffer (Seeley et al., 1992) and shaken at 75 rpm for 30 min at room
temperature. At the end of this period, cordycepin (Sigma) was added to a
final concentration of 0.6 mM and the samples were vacuum infiltrated for 45
s at 18 in Hg. Samples were then harvested from the 250-mL flask at regular
time intervals starting from time zero up to 16 h, blotted, and immediately
frozen in liquid nitrogen. Total RNA isolation and quantitative RT-PCR with
gene-specific primers were performed as described previously (Banerjee
et al., 2006a).
For StBEL5 native transcript analysis, the BEL5 gene-specific primers
5#-GGGAGATTTTGGAAGGTTTG-3# and 5#-TCAAATTGGGTCCTCCGACT-3#
were used. For quantitative analysis of StBEL5 cds and StBEL5 full-length
transcripts (Fig. 5, A and B), the primers and RT-PCR conditions described
previously were used (Banerjee et al., 2006a).
Translation Assay
The CaMV 35S promoter in pBI221 (Clontech) was used for translation
assays with the three combinations of UTR fusions to the GUS reporter
construct (Fig. 6A). The reporter alone in pBI221 was used as a translation
control. A CaMV 35S promoter-driven luciferase (LUC) construct, 35S-LUC
(obtained from Dr. Y. Takahashi, Department of Biological Sciences, Graduate
School of Science, University of Tokyo), was used as an internal control. Fully
expanded leaves from 3- to 4-week-old tobacco plants were excised, placed in
K3 basal medium (Kao and Michayluk, 1975) supplemented with 0.4 M Suc,
0.25% (w/v) cellulases (Karlan Research Products), and 0.05% (w/v) macerases (Calbiochem), and incubated overnight at 28°C. The protoplasts were
then filtered through sterile cheesecloth into a Babcock bottle and centrifuged
for 10 min at 1,000 rpm. Protoplasts were collected from the bottleneck
area and washed once in K3 medium with 0.4 M Suc and resuspended in
K3 medium containing 0.4 M Glc to a final concentration of 4 3 106 protoplasts mL21.
For each transfection analysis, 700 mL of tobacco protoplasts (prepared as
described above) was mixed with 30 mL of 2.0 M KCl and plasmid DNA in an
electroporation cuvette with a 0.4-cm electrode gap. The plasmid DNA was a
mixture of 5.0 mg of the GUS reporter construct and 0.5 mg of the 35S-LUC
construct as an internal control. After electroporation (voltage = 170 V,
capacitance = 125 mF; Gene Pulser Transfection Apparatus [Bio-Rad]), 4.0
mL of Murashige and Skoog (1962) basal medium was added, and the
protoplasts were incubated in the dark at room temperature for 48 h before
conducting GUS and LUC activity assays. Transfections were performed three
times for each construct.
LUC assays were performed by injecting 100 mL of LUC substrate
(Promega) into 20 mL of extract and measuring the emitted photons for 15 s
in a TD-20 luminometer (Turner Designs). Fluorometric GUS assays were
performed as described (Jefferson, 1987). A fluorescence multiwell plate
reader (Fluoroskan II; MTX Labs) was used to measure GUS activity at 365 nm
(excitation) and 455 nm (emission). Each sample was measured three times for
both LUC and GUS activity. A relative value for GUS expression was obtained
by dividing the GUS activity by the specific LUC activity. Relative activities
were calculated from three transfection replications and are presented as
means 6 SE.
ACKNOWLEDGMENT
Thanks to Brian A. Campbell for his valuable technical assistance.
Received July 7, 2009; accepted September 21, 2009; published September 25,
2009.
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