A BELL1-Like Gene of Potato Is Light Activated and
Wound Inducible1[C][OA]
Mithu Chatterjee, Anjan K. Banerjee, and David J. Hannapel*
Department of Horticulture, Iowa State University, Ames, Iowa 50011–1100
BELL1-like transcription factors interact with their protein partners from the KNOTTED1 family to bind to target genes and
regulate numerous developmental and metabolic processes. In potato (Solanum tuberosum), the BELL1 transcription factor StBEL5
and its protein partner POTH1 regulate tuber formation by affecting hormone levels. Overexpression of StBEL5 in transgenic lines
produces plants that consistently exhibit enhanced tuber formation, and the mRNA of this gene moves through phloem cells in a
long-distance signaling pathway regulated by photoperiod. Whereas photoperiod mediates the movement of StBEL5 RNA,
activation of transcription of the StBEL5 gene in leaves is regulated by white light, regardless of photoperiod or light intensity.
Illumination with either red or blue light induces the StBEL5 promoter, whereas far-red light had no effect. As expected, the StBEL5
promoter harbors numerous conventional light-responsive cis-acting elements like GT1, GATA, and AT1 motifs. Deletion constructs were analyzed to determine what sequences are involved in light activation. Transcriptional activity was also mediated by
wounding on stems, insect predation on leaves, and photoperiod in stolons. These results demonstrate that StBEL5 gene activity in
the leaf is correlated with wavelengths optimal for photosynthesis. The number of factors that affect the StBEL5 promoter supports
the premise that the BELL1-like genes play a role in a wide range of functions.
Homeobox-containing genes encode transcription
factors that play an important role in plant morphogenesis and development. The first homeotic gene was
discovered in Drosophila and subsequently isolated from
distinct evolutionary groups like fungi, plants, and animals (Chan et al., 1998). The protein encoded by homeobox genes contains a conserved DNA-binding domain,
the homeodomain consisting of three a-helices (Desplan
et al., 1988; Otting et al., 1990; Laughon, 1991). The first
plant homeobox gene identified in maize (Zea mays),
KNOTTED1, is involved in formation and maintenance
of the shoot apical meristem (Vollbrecht et al., 1991;
Kerstetter et al., 1997; Hake et al., 2004). Another plant
homeobox gene family closely related to KNOTTED1
is the BELL1 family (Quaedvlieg et al., 1995; Reiser et al.,
1995; Chan et al., 1998). KNOTTED1-like and BELL1like genes belong to the TALE superclass of transcription
factors characterized by a three-amino-acid loop extension present between the first and second a-helices
of the homeodomain (Bürglin, 1997). In Arabidopsis
(Arabidopsis thaliana), BELL1 (BEL1) regulates ovule development and the specification of outer and inner in1
This work was supported by the National Science Foundation in
the Division of Integrative Organismal Biology (award no. 0344850).
* 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.107.105924
teguments (Reiser et al., 1995). Molecular and genetic
analyses revealed that BELL1, along with the carpeldetermining homeotic gene AGAMOUS, participates
in several distinct aspects of ovule morphogenesis (Ray
et al., 1994; Reiser et al., 1995; Gasser et al., 1998; Western
and Haughn, 1999). Subsequent studies of BELL1-like
genes indicate that they are ubiquitous in the plant
kingdom. The apple (Malus domestica) BELL1-like homeodomain gene, MDH1, plays an important role in
growth, fertility, and development of carpels and fruit
shape (Dong et al., 2000). Two Arabidopsis null mutants
of the BELL1-like genes PENNYWISE and POUNDFOOLISH disrupt the transition from vegetative to floral development (Smith et al., 2004). Tobacco (Nicotiana
tabacum) plants overexpressing BELL1 of barley (Hordeum vulgare) were dwarf with multiple shoots and
exhibited malformed leaves and flowers (Müller et al.,
2001). Several BELL1-like cDNAs have been identified
in potato (Solanum tuberosum) and one, StBEL5, is involved in tuber development by affecting hormone
levels (Chen et al., 2003, 2004). Recently, a rice (Oryza
sativa) homolog, OsBIHD1, was identified that functions in disease resistance and pathogen defense (Luo
et al., 2005). Clearly, BELL1-like genes function in a wide
range of processes in plants.
In potato, using the KNOTTED1-like protein POTH1
as bait in the yeast two-hybrid system, seven BELL1like proteins were identified (Chen et al., 2003). Overexpression of one of the BELLs, StBEL5, and POTH1 in
transgenic lines produces plants that consistently exhibit enhanced rates of tuber growth (Chen et al., 2003;
Rosin et al., 2003). In these plants, RNA levels for GA
20-oxidase1 were reduced in stolons and leaves, respectively. This reduction in GA 20-oxidase1 RNA accumulation coincides with a reduction in active GA levels
Plant Physiology, December 2007, Vol. 145, pp. 1435–1443, www.plantphysiol.org Ó 2007 American Society of Plant Biologists
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Chatterjee et al.
and an increase in tuberization. During tuberization,
the levels of GA1 in swollen stolons decreases due to
the activity of key regulatory enzymes in the GA biosynthetic pathway (Xu et al., 1998a; Kloosterman et al.,
2007). Similarly, overexpression mutants of other KNOX
genes from tobacco (NTH15) and rice (OSH1) also exhibited reduced activity of GA 20-oxidase1 and decreased
accumulation of GA1 (Tamaoki et al., 1997; Kusaba et al.,
1998a, 1998b; Tanaka-Ueguchi et al., 1998).
During tuberization, POTH1 appears to form a tandem
complex with its partner StBEL5 to regulate development (Chen et al., 2003, 2004). Detailed studies indicated that POTH1 and StBEL5 must interact to bind
the target elements and to affect transcription of the
GA 20-oxidase1 gene (Chen et al., 2004). These results
demonstrated that both protein partners are essential
in binding the cis-element to modulate transcription.
Protein-protein interaction has also been demonstrated
between members of the BELL1 and KNOX families in
Arabidopsis (Bellaoui et al., 2001; Kanrar et al., 2006;
Viola and Gonzalez, 2006), barley (Müller et al., 2001),
and maize (Smith et al., 2002).
To gain more insight into the factors that regulate the
activity of the BELL1 genes of potato, in this study, the
promoter of StBEL5 has been characterized. Despite
the importance of BELL1-like genes in plant biology,
very little is known about the regulation of their transcription. This is particularly significant in light of a recent
report documenting the correlation of photoperiodmediated movement of the full-length mRNA of StBEL5
with tuber formation (Banerjee et al., 2006a). Because
StBEL5 may be involved in controlling tuberization and
this stage of development is regulated by photoperiod
(Rodriguez-Falcon et al., 2006), this study focuses on the
light regulation of the StBEL5 promoter. Our results
indicate that the StBEL5 promoter is activated by light
and is insensitive to photoperiod in the leaf and that
it harbors numerous conventional light-responsive cisacting elements like GT1, GATA, and AT1 motifs. Deletion constructs were analyzed to determine what
sequences are involved in light activation. Promoter
activity was also mediated by wounding on stems,
insect predation on leaves, and photoperiod in stolons.
The number of factors that affect the StBEL5 promoter
supports the premise that the BELL1-like genes play a
role in a wide range of functions.
1997) and AT1 motifs (Fig. 1B). The G-box LRE was
identified at positions 21,030, 2693, 327, and 94. Most
of the LREs were present in multiple copies. For example, nine GATA boxes, 11 GT1 boxes, and four AT1
motifs were identified in this 2.29-kb upstream region.
A careful analysis revealed the presence of a large number of light elements within 500 bp of the promoter
region near the transcription start site (10 were identified). Clustering of LREs within this region suggests
that the combination of these elements may function
as a minimal light-regulatory region. The GATA boxes
tended to be distributed in the flanking regions of the
promoter, whereas the GT1 boxes are preponderate in
the middle region. No LREs were identified in the intron
RESULTS
cis-Regulatory Elements of the StBEL5 Promoter
To elucidate how the promoter of StBEL5 regulates
transcription, the 2.29-kb upstream region (Fig. 1A) was
screened for cis-acting regulatory elements using the
software PLACE (Higo et al., 1999) and the PlantCare
database (Lescot et al., 2002). We observed the presence of several putative cis-acting light-regulatory elements (LREs), including GT1 and GATA boxes (Giuliano
et al., 1988; Terzaghi and Cashmore, 1995; Guilfoyle,
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Figure 1. StBEL5 upstream regions. A, Schematic diagram of the structure consisting of promoter, 5#-UTRs, and an intron. B, DNA sequence
represents the StBEL5 upstream region plus 150 nucleotides of the
5#-UTR. Nucleotide sequences representing potential LREs are highlighted: GATA box, gray box; GTI motifs, white box; and AT1 motifs,
underlined. The divided 5#-UTR is bracketed and italicized and is
flanking the intron, which is designated by light letters from nucleotides
97 to 299 (B). Note: The sequence from 22,002 to 21,938 is a cloning
adaptor.
Plant Physiol. Vol. 145, 2007
BEL5 Promoter Activity
Figure 2. Effect of wavelength on the activity of the StBEL5 promoter.
For light induction assay, in vitro transgenic potato plants containing
P-StBEL5 were grown for 15 d in white light (16 h light, 8 h dark), 15 d in
the dark, or 14 d in the dark followed by blue-, red-, far-red-, or whitelight irradiation for 24 h. Several shoot tips, approximately 1.0 cm in
length, were harvested for the GUS assays. GUS activity is expressed in
nanomoles of 4-methylumbelliferone (4-MU) produced per hour per
microgram of protein. Data represent the mean 6 SD of GUS activity
measured with three replicates. For white light, in vitro cultures were
incubated at a light intensity of 40 mmol m22 s21 cool-white fluorescent
light at 27°C in a Percival incubator. A single incandescent bulb was
used for the far-red light treatment (5 mmol m22 s21) and a single
fluorescent tube was screened with select filters to produce blue and
red wavelengths (8 mmol m22 s21).
(Fig. 1B, nucleotides 97–299). In addition to LREs, software analyses also identified GA-responsive elements
like P box, TATC box, and WRKY71OS (Gubler and
Jacobsen, 1992; Lanahan et al., 1992; Zhang et al., 2004),
and several wound-response elements like W box,
WUN motif, and G boxes (Siebertz et al., 1989; Rushton
and Somssich, 1998; Rushton et al., 2002).
Light-Activated Expression of the StBEL5 Promoter
Because StBEL5 appears to be involved in controlling
tuberization and this stage of development is regulated by photoperiod (Rodriguez-Falcon et al., 2006),
the light regulation of the StBEL5 promoter was evaluated. Analysis of transcription was performed on
transformed potato plants containing the P-StBEL5 promoter construct. The design and characterization of
this promoter:GUS construct was previously reported
(Banerjee et al., 2006a). To evaluate the effect of light on
the StBEL5 promoter, GUS activity was determined using in vitro-grown shoot tips of transgenic potato plants.
Promoter activity was examined in samples grown
under three different light conditions, 15 d light (16 h
light, 8 h dark), 15 d dark, or 14 d dark followed by 24 h
exposure with various wavelengths of light (Fig. 2).
Each set of experiments was conducted with three independent lines. Here, data of one representative line
Plant Physiol. Vol. 145, 2007
are presented. The lines containing the P-StBEL5 construct showed lower GUS activity when grown under
dark conditions and activity increased 2- to 3-fold
upon illumination with white light (Fig. 2). Light induction was also observed after 2 h of light treatment
following the dark period (data not shown). For evaluation of specific wavelengths, after the dark treatment, in vitro plants were exposed to 24 h of blue light
(8 mmol m22 s21), red light (8 mmol m22 s21), or far-red
light (5 mmol m22 s21). With blue and red light, a 2- to
3-fold increase in GUS activity was observed, whereas
exposure to far-red light induced only a modest increase (Fig. 2). Because a short-day (SD) photoperiod
facilitates movement of StBEL5 mRNA and induces
tuber formation, the effect of photoperiod on StBEL5
promoter activity was assayed (Fig. 3). GUS expression in leaves and petiole samples (Fig. 3A) harvested
from plants grown under either long-day (LD; 16 h
light, 8 h dark) or SD (8 h light, 16 h dark) conditions
was evaluated. Transcriptional activity was enhanced
under both photoperiod conditions. Both petioles and
leaves exhibited greater levels of StBEL5 promoter activity under LD conditions (Fig. 3B). This experiment,
however, does not separate the effects of photoperiod
from light quantity because LD plants are exposed to a
greater total fluence rate. To test photoperiod effects,
SD (8 h light, 16 h dark) and SD plus night break (7.5 h
light with a 0.5-h night break) conditions for 12 d were
utilized. The night break simulates LD (or short night)
and delivers an equivalent irradiation quantity. In this
experiment, photoperiod had no effect on promoter
activity in leaves or petioles (Fig. 3C). To determine
whether light quantity is controlling promoter activity,
P-StBEL5 plants were exposed to LD (16 h light, 8 h
dark) conditions in a growth chamber under a fluence
rate of 60, 200, or 400 mmol m22 s21 for 12 d. No difference in promoter activity was observed in petioles
and leaf veins upon exposure to any of the three fluence
rates (Fig. 3D).
To define the minimal light regulatory region, two deletion constructs, designated P1-StBEL5 and P2-StBEL5,
were designed, fused to a GUS sequence (Fig. 4A) and
transformed into potato (Banerjee et al., 2006b). P1StBEL5 is composed of 1,052 nucleotides of promoter
sequence (extending from nucleotide 21,052 to the start
of the untranslated region [UTR]) plus 96 nucleotides
of the 5#-UTR, whereas P2-StBEL5 is composed of 272
nucleotides of the downstream promoter sequence (extending from nucleotide 2272 to the start of the UTR)
plus 96 nucleotides of the 5#-UTR. There were no observable differences in leaf, stem, root, or stolon expression between constructs with or without the intron,
and this sequence was not included in constructs P1
and P2. Both constructs exhibited light induction when
exposed to 24 h of white-light treatment (Fig. 4B). The
P2 construct apparently contained sufficient LREs (three
GATA, two GT1, and one AT1) to confer light induction. The expression driven by this smaller fragment,
however, is more than 10-fold lower than the lightdriven activity of P-StBEL5. P1-StBEL5 induction was
1437
Chatterjee et al.
Figure 3. Effect of photoperiod on StBEL5 promoter
activity. GUS expression was measured in leaf (lamina and veins) and petiole samples (A) grown under
either LD (16 h light, 8 h dark) and SD (8 h light, 16 h
dark) conditions (B), SD (8 h light, 16 h dark) and
night break (7.5 h light, 16 h dark with a 0.5-h light
night break) conditions for 12 d each (C), or under
three fluence rates of 60, 200, or 400 mmol m22 s21
for 12 d (D). The data represent the mean 6 SD of
GUS activity measured with three replicates. GUS
activity (B and C) was performed on soil-grown plants
incubated at a light intensity of 100 mmol m22 s21
with cool-white fluorescent bulbs plus incandescent
light at 22°C light period/18°C dark period. The same
environmental conditions were used for the light
intensity experiment (D) except for adjustment of the
fluence rate. [See online article for color version of
this figure.]
about 2-fold less than P-StBEL5 activity. Histochemical
analysis of leaves of P1 was consistent with this decrease in GUS activity (Fig. 5A). A faint GUS reaction
was observed in the primary vein of P1 leaves (Fig. 5A,
arrow). The relative promoter activities of the three
constructs tested here are correlated to the number of
LREs identified in each construct. Accounting for GATA,
GT1, and AT1 elements only, P-StBEL5, P1-StBEL5, and
P2-StBEL5 contained 24, 12, and six total elements, respectively.
Regulation of StBEL5 Promoter Activity in
Underground Organs
Previous work has shown that, in addition to light
activation in veins and petioles of leaves, StBEL5 promoter activity was observed consistently in stolons from
both tuberizing and nontuberizing plants, in newly
formed tubers, and in roots (Banerjee et al., 2006a). To
examine what sequences contributed to promoter activity in the dark, the three promoter constructs previously described were analyzed for activity in three
underground organs formed on potato: roots, stolons,
and tubers. Histochemical analyses of roots (Fig. 5B)
and stolons/tubers (Fig. 5, C–G) indicated that expression of GUS was incrementally reduced in the P1 and P2
constructs. Only a very low level of GUS staining was
observed in roots of P2 transgenic lines (Fig. 5B),
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whereas no GUS expression was observed in stolons
and newly formed tubers from these lines (Fig. 5, F and
G). Although GUS staining was diffuse in tubers from P
transgenic lines, promoter activity was greatest in
vascular connections of newly formed tubers (Fig. 5D,
arrows), in internal and external phloem strands (Fig.
5E, in and ex, respectively), and in the peridermal layer
(Fig. 5E, pe). Activity of the StBEL5 promoter coincides
with the region of most active cell growth in a new
tuber, the perimedullary region between the pith and
the phloem (Xu et al., 1998b).
To investigate further the mechanism for this activation in the dark, promoter activity was assayed in
tuberizing stolons grown on SD plants and nontuberizing stolons from LD plants. No signal was detected
in stolons from plants grown for 6 d in a growth chamber under LD conditions (Fig. 6). The overall pattern of
activity showed no trend through 12 d of LD exposure.
Activity in stolons from SD plants, however, exhibited
a steady increase from day 6 through day 12. This
activity corresponds to both the onset of tuber formation
and the gradual accumulation of mRNA for StBEL5
(Chen et al., 2003).
Wounding Effects
Previous analyses of a transverse stem section collected from the internodal regions of transgenic plants
Plant Physiol. Vol. 145, 2007
BEL5 Promoter Activity
Figure 4. A, Schematic diagram of the deletion constructs of StBEL5 promoter fused to a GUS reporter
gene. The upstream region is indicated by the gray
box, and the 5#-UTR includes sequence from 1 to 96
and 300 to 354 and is interrupted by an intron from
nucleotide 97 to 299 (diagonal-hatched box). Constructs P1 and P2 do not include any intronic sequence. B, Transgenic plants expressing each of the
three constructs P-StBEL5, P1-StBEL5, and P2-StBEL5
exhibited light-mediated induction following a 24-h
white-light treatment. In vitro plants were grown
under light conditions (L), 15 d in dark (D), or 14 d
in dark followed by white light (WL) irradiation for
24 h. GUS activity was assayed on in vitro plantlets
incubated at a light intensity of 40 mmol m22 s21 coolwhite fluorescent light at 27°C in a Percival incubator.
The lower scale of promoter activity in this experiment
(compare to Fig. 3, B–D) is probably due to the quality
of light provided by the Percival unit. No incandescent
bulbs were used in this analysis.
harboring the P-StBEL5 construct showed that GUS is
not expressed in stem cross sections (Banerjee et al.,
2006a). However, we consistently observed that the cut
ends of stems exhibited GUS activity (Fig. 7A). Stem
samples of transgenic plants containing the P-StBEL5
and P1-StBEL5 constructs exhibited a high level of GUS
activity when subjected to mechanical injuries (Fig. 7A),
whereas P2-StBEL5 transgenic lines exhibited very little, if any, wound response (Fig. 7B). According to software analyses, overall, the StBEL5 promoter sequence
contains 12 G boxes, 21 WUN motifs, and nine W boxes.
In the P2 construct, this number drops to two, three,
and one, respectively. When leaves were subjected to
the same mechanical injury, no GUS expression was
observed in P lines. No promoter activity was observed
even when leaves were cut into small pieces with a
razor blade (Fig. 7C). The leaves, however, exhibited a
response to insect attack. Those leaves infested with
western flower thrips (Frankliniella occidentalis) showed
promoter activity coincident to the area of infection and
at the junction of primary and secondary veins (Fig.
7D, arrows). GUS expression was also observed in the
mesophyll region of leaves undergoing senescence
(data not shown). The presence of wound-response
elements like W box, WUN motif, and G boxes in the
promoter supports the premise that StBEL5 transcription is activated by wound- and pest-induced pathways.
DISCUSSION
Because of the importance and ubiquity of the BELL1like family (Hake et al., 2004), the lack of information
on promoter activity for any of these genes, and the
reputed role of full-length StBEL5 mRNA as a mobile
signal for tuber formation (Banerjee et al., 2006a),
analysis of its promoter activity would be highly informative. Of particular interest is the source of this
Figure 5. Expression analysis of three deletion constructs of the StBEL5 promoter fused to the GUS reporter gene in leaves (A), roots (B), and newly formed
tubers (C–G) of transgenic potato plants containing
construct P (C–E), P1 (C), or P2 (F–G). These constructs
were described in Figure 4A. Leaves were from plants
grown under LD conditions with white light and the
roots and tubers were harvested from plants grown for
10 d of SD conditions at a light intensity of 100 mmol
m22 s21 with cool-white fluorescent bulbs plus incandescent light at 22°C. D, E, and G, Internal longitudinal sections. The size bar in E represents 0.1 cm.
Arrows in D and E indicate phloem strands. in, Internal
phloem; ex, external phloem; pe, peridermal tissue.
Anatomy of the newly formed tubers is based on the
careful study performed by Reeves et al. (1969).
Plant Physiol. Vol. 145, 2007
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Chatterjee et al.
Figure 6. Promoter activity of StBEL5:GUS in stolons from plants grown
under SD (8 h light, 16 h dark) or LD (16 h light, 8 h dark) days. GUS
quantification was performed on stolon tips harvested from transgenic
lines containing the P-StBEL5 construct. Stolon tips (approximately 1.0 cm
in length) were harvested after 6, 8, and 12 d from whole plants grown at a
light intensity of 100 mmol m22 s21 with cool-white fluorescent bulbs plus
incandescent light at 22°C. ND, Not detected. Three replicates were
averaged for each harvest and error bars are indicated.
mobile RNA and how the BEL5 promoter is regulated
in relation to light and the creation of a strong underground sink, the tuber. BELL1-like transcription factors control numerous facets of plant development and
metabolism, but their function in light perception and
signal transduction is largely unknown. ATH1 of Arabidopsis is one example of a BELL1-like protein whose
expression is tightly regulated by exposure to light
(Quaedvlieg et al., 1995). Dark-grown seedlings of
photomorphogenic mutants possessed elevated ATH1
mRNA levels in comparison with etiolated wild-type
seedlings. Genetic analyses have demonstrated that
ATH1 may be an important downstream component
of the DET1 and COP1 signal transduction pathways
and involved in the expression of light-inducible genes
and photomorphogenic control. Here, we have focused
on the role that light plays in regulating the transcription of a BELL1-like gene from potato, StBEL5.
The promoter sequence of StBEL5 contains several
LREs and the presence of these elements was correlated with light elicitation (Fig. 4). Dark-grown plants
exhibited enhanced promoter activity within 2 h of exposure to white light (data not shown). This enhanced
activity was wavelength dependent, with blue and red
light eliciting significant activation (Fig. 2). These results imply that promoter activity may be selectively
mediated by phytochrome and/or blue-light receptors. Perception and transduction of light are achieved
by at least two principal groups of photoreceptors, phytochromes and cryptochromes (for review, see Jiao et al.,
2007). Phytochromes are red/far-red light-absorbing
receptors encoded by a gene family of five members
(phyA–phyE) in Arabidopsis. Cryptochrome 1, cryptochrome 2, and phototropin are the blue-light receptors
that have currently been identified. The phototropins
1440
primarily regulate processes optimizing photosynthesis,
whereas transcriptional and developmental changes
are attributed to the phytochromes and the cryptochromes. Blue light (390–500 nm) elicits a variety of
physiological responses in plants, including four that
maximize photosynthetic potential. These include phototropism, light-induced opening of stomata, chloroplast
migration in response to changes in light intensity, and
solar tracking (Kinoshita et al., 2001; Briggs and Christie,
2002).
Cryptochromes interact genetically with multiple
phytochromes (Neff et al., 2000). For example, phytochrome B and cryptochrome 2 have been shown to
tightly colocalize in vivo, bind in vitro, and interact in
the control of flowering time, hypocotyl elongation,
and circadian clocks (Mas et al., 2000). Blue- and redlight fluence rates as low as 8 mmol m22 s21 were as
effective as white-light treatments of 40 mmol m22 s21
in inducing the StBEL5 promoter (Fig. 2). In addition,
the red and blue filters delivered light at 650 and 450
nm, both of which are well within the spectral range
Figure 7. Wound response of the StBEL5 promoter. A, Stem segments of
transgenic plants containing P1-StBEL5 subjected to mechanical injury
with a razor blade (left-side segment) or a forceps (right-side segment)
and immediately incubated in GUS buffer as described in ‘‘Materials
and Methods.’’ The middle segment is an excised stem portion that was
not wounded. Similar results were obtained from transgenic lines
harboring the P-StBEL5 construct. B, Wounded stem segments from
transgenic lines harboring the P2-StBEL5 construct. C, Leaves from
transgenic lines of P-StBEL5 subjected to mechanical wounding. D,
Leaf from transgenic plants of P-StBEL5 infested with the western flower
thrip. Note sites of insect feeding (arrows).
Plant Physiol. Vol. 145, 2007
BEL5 Promoter Activity
for both photosynthetic activity and light absorption
(Taiz and Zeiger, 2006).
Although there are examples where the minimal
light-driven regulatory region on a promoter can be as
short as 52 bp (Schafer et al., 1997; Martinez-Hernandez
et al., 2002), several studies have confirmed that the minimal light-driven region is generally found within 250 bp
upstream from the transcriptional start site (Terzaghi
and Cashmore, 1995). Deletion analysis of the StBEL5
upstream region revealed that the construct containing
272 nucleotides of promoter plus 96 nucleotides of the
5#-UTR P2-StBEL5 was sufficient for light-mediated
activation (Fig. 4). It is possible that the combinatorial
interaction of the three GATA, two GT1, and one AT1
elements in this sequence is sufficient to participate in
light induction, allowing the P2-StBEL5 construct to act
as a minimal light regulatory sequence.
But how can the activity of the StBEL5 promoter that
occurs in stolon tips and tubers in the dark (Fig. 5, C–E)
be reconciled with light induction? Similar to the light
response, this activity in the dark is progressively reduced in the P1 and P2 constructs (Fig. 5, B and C, and
F and G). Photoperiod only affects promoter activity in
stolon tips (Fig. 6). In this system, white-light exposure, regardless of intensity or photoperiod, activated
the BEL5 promoter to its greatest levels in leaf veins
and petioles. In response to SDs, phloem transport of
full-length StBEL5 mRNA is enhanced, delivering this
mRNA to stolon tips (Banerjee et al., 2006a). StBEL5
promoter activity in stolon tips increases in response to
SDs as the tuber develops (Fig. 6). BEL5 and its Knox
partner bind to a specific tandem TTGAC motif on their
target promoters to affect transcription (Chen et al.,
2004). Such a double motif is present on the promoter
of StBEL5 on opposite strands beginning at nucleotide
2820 (GTCAATGCTTGAC; Fig. 1B, dotted line). Based
on these observations, it is feasible that StBEL5 protein
(with its Knox partner) autoregulates its own promoter to transduce a light-mediated signal from the
leaf to the underground storage organ, the tuber. This
would be consistent with the observations that SDs
activate the StBEL5 promoter in stolon tips (Fig. 6) and
that the P2 construct (missing the putative BEL5/Knox
motif) is not active in stolons and newly formed tubers
(Fig. 5, F and G).
The StBEL5 promoter appears to be regulated by a
diverse and complex array of factors. A wound response was observed on stems, but not leaves, whereas
insect predation activated the promoter in leaves. Promoter activity in roots, stolons, and tubers is subject to
a process active in the dark, whereas leaves respond to
light. These results are consistent with the fact that the
BELL1 family, along with its respective Knox partners,
is involved in a number of developmental and metabolic processes. Clearly, BELL1-like genes function in
more than just ovule and inflorescence development.
For example, Brevipedicellus, a KNOTTED1-like transcription factor of Arabidopsis, regulates several genes
involved in lignin biosynthesis, an important biochemical pathway of secondary cell wall growth (Mele et al.,
Plant Physiol. Vol. 145, 2007
2003). A BELL1-like gene from rice, OsBIHD1, was identified that functions in disease resistance and pathogen
defense (Luo et al., 2005).
Of the numerous signaling pathways mediated by
light or photoperiod, the one most similar to the induction of tuber formation is flowering (for review, see
Rodriguez-Falcon et al., 2006). Both processes are controlled by the action of phytochrome A, phytochrome
B, and blue-light receptors (Endo et al., 2007). It has
been clearly established that phytochrome B plays a
pivotal role in regulating tuber formation (Jackson et al.,
1996; Jackson and Prat, 1996). Both pathways involve
the mobilization of a photoperiod-mediated signal that
moves long distance to activate a newly forming sink
(Banerjee et al., 2006a; Lin et al., 2007). There are important differences, however. During flowering, the signal moves from a light organ, the leaf, to another organ
in the light, the shoot tip. Potato tuberization is unusual in that it involves a signal that arises in the light
and is transported to an underground organ in the dark,
the stolon tip. In this light-to-dark model, white light
activates transcription in the leaf in coordination with
photosynthesis. Both blue and red light, in the optimal
ranges for the action spectrum of photosynthesis, activate the StBEL5 promoter (Fig. 2). Under the LDs of
the growing season, both StBEL5 transcripts and photoassimilate accumulate in the leaves, ready for delivery.
Late in the season, as days shorten, StBEL5 mRNA is
transported to the stolon tip, translated, and imported
into the nucleus. In tandem with its Knox partner,
StBEL5 could then target genes that activate cell division and expansion in the subapical region of the stolon
tip. Adequate supplies of sugar in the leaf are then
translocated to the newly formed sink for the synthesis
of starch in the tuber. In this way, the trigger for the
development of tuber formation, a bioenergic-expensive
process, is coordinated with the production of sugars
in photosynthesis.
MATERIALS AND METHODS
Plant Material and Growth Condition
To assess photoperiod effects, transformation was implemented on the
photoperiod-responsive potato (Solanum tuberosum) subsp. andigena (Banerjee
et al., 2006b). In SD-adapted genotypes like subsp. andigena, SD photoperiods
(,12 h light) are required for tuber formation, whereas under LD conditions
no tubers are produced. In vitro transgenic potato plants were grown at 27°C
with a photoperiod of a 16-h-light (fluence rate of 40 mmol m22 s21) and 8-hdark cycle in a growth chamber (Percival Scientific). Soil-grown plants were
grown at 22°C with a photoperiod of 16-h-light (fluence rate of 100 mmol m22 s21)
and 8-h-dark cycle for LD conditions and, for a SD photoperiod, plants were
transferred for 14 to 16 d under a 16-h-dark and 8-h-light cycle in a growth
chamber. For light induction experiment, one set of in vitro-grown plants were
kept in a 16-h-light and 8-h-dark cycle for 15 d, one set in dark for 15 d, and
one set in dark for 14 d followed by light treatment for 24 h. For dark treatment, plants were transferred to a dark incubator maintained at 27°C.
Light Source and Energy Measurements
For various light wavelength treatments of transgenic in vitro plants, the
light filters from Carolina Biological Supply (CBS) red 650, CBS far-red 750,
and CBS blue 450 were used. The source of light from the plant material was
1441
Chatterjee et al.
adjusted to yield 8 mmol m22 s21 (red and blue) and 5 mmol m22 s21 (far red) of
uniform irradiation as measured by the LI-189 radiometer (LI-COR). White
light was provided from a bank of fluorescent tubes with a fluence rate of
40 mmol m22 s21.
Promoter Deletion Constructs and Transformation
of Potato
For the two deletion constructs P1-StBEL5 and P2-StBEL5 (Fig. 4A), genomic
fragments were amplified using PCR with primers 5#-GCTCTAGAAACCTGTGGTCGGAGTGGAC-3#, 5#-GCTCTAGACACTCCACAATCTACCGAAACA-3#, and 5#-TACCCGGGATGTAACTAACTGTCTATCTTCGGG-3#. For
amplification of the P1-StBEL5 product, the PCR cycle was as follows: 3 min
at 94°C; 35 cycles (30 s at 94°C; 30 s at 58°C; 1.20 min at 68°C); followed by 68°C
for 10 min. For the P2-StBEL5 product, PCR reaction was performed as follows: 3 min at 94°C; 35 cycles (30 s at 94°C; 30 s at 58°C; 30 s at 68°C); with a 10-min
extension step at 68°C. The amplified promoter fragments were cloned in
pBI101 vector (Jefferson et al., 1987) and mobilized to Agrobacterium tumefaciens
strain GV2260 by chemical transformation (An et al., 1988). These deletion
constructs were then transferred to potato subsp. andigena employing an
Agrobacterium-mediated transformation protocol (Banerjee et al., 2006b).
Kanamycin-resistant and GUS-positive transgenic plants were selected for
further analysis. The isolation and characterization of the P-StBEL5 construct
was previously reported (Banerjee et al., 2006a). The 2.29-kb upstream fragment was sequenced and analyzed with software PLACE, plant cis-acting
regulatory elements (Higo et al., 1999), and PlantCARE (Lescot et al., 2002).
Histochemical and Fluorometric Analyses
Expression of the GUS reporter gene 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/mL X-gluc (5-bromo-4-chloro-3-indolylb-D-GlcUA). Samples were cleared with 100% ethanol and photographed
employing a Nikon COOLPXX995 digital camera.
Frozen tissue samples were homogenized using 100 mL of chilled extraction buffer containing 50 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA,
0.1% sarcosine, 10 mM 2-malic enzyme. Samples were then centrifuged at
13,000 rpm for 15 min, at 4°C. The supernatant obtained was used for protein
quantification (Bradford, 1976). Extracts containing approximately 10 mg of
protein were aliquoted and their volume made up to 50 mL with extraction
buffer containing 2.0 mM 4-methylumbelliferyl-b-D-glucuronide and incubated at 37°C for 16 h. The reaction was stopped by adding 200 mL of stop
buffer (0.2 M Na2CO3). The relative fluorescence was measured using a fluorometer (FluroMax-2) with an excitation wavelength of 355 nm and an emission wavelength at 460 nm. The specific activity of GUS was calculated using a
calibration curve for 4-methylumbelliferone.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession number EU200938.
ACKNOWLEDGMENT
We thank Dr. Suqin Cai for her assistance with photodocumentation.
Received July 19, 2007; accepted September 26, 2007; published October 5,
2007.
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