Breakthrough Technologies
A Tightly Regulated Genetic Selection System with
Signaling-Active Alleles of Phytochrome B1[OPEN]
Wei Hu and J. Clark Lagarias*
Department of Molecular and Cellular Biology, University of California, Davis, California 95776
ORCID IDs: 0000-0001-9602-1428 (W.H.); 0000-0002-2093-0403 (J.C.L.).
Selectable markers derived from plant genes circumvent the potential risk of antibiotic/herbicide-resistance gene transfer into
neighboring plant species, endophytic bacteria, and mycorrhizal fungi. Toward this goal, we have engineered and validated
signaling-active alleles of phytochrome B (eYHB) as plant-derived selection marker genes in the model plant Arabidopsis
(Arabidopsis thaliana). By probing the relationship of construct size and induction conditions to optimal phenotypic selection,
we show that eYHB-based alleles are robust substitutes for antibiotic/herbicide-dependent marker genes as well as surprisingly
sensitive reporters of off-target transgene expression.
The promise of agricultural biotechnology has been
tempered by political, economic, food allergy, intellectual property, and ecological concerns that have led to
restrictions on more widespread acceptance of genetically modified organism technology. Particularly acute
in Europe and Asia, such concerns have helped sustain
regulatory barriers that restrict adoption of genetically
modified organism approaches to improve agronomic
performance of key food crop species consumed by
humans, e.g. rice (Oryza sativa), wheat (Triticum aestivum),
and leafy vegetable crops. The human population is estimated to reach 10 billion before the end of this century,
while the amount of arable land is not expected to increase proportionately (Borlaug, 2007; United Nations,
2011). These issues are further compounded by climate
change that threatens agronomic performance of plant
varieties suited to their local environment at rates faster
than conventional breeding can compensate (Long and
Ort, 2010). Climate change is further exacerbated by the
reliance on fossil fuels for energy and the concomitant
release of greenhouse gases (O’Neill et al., 2010). The toll
of rising costs of energy and increasing urbanization will
elevate food prices, fueling famine, disease, and political
unrest throughout the world. While there is no easy solution to these problems, it is inevitable that agricultural
1
This work was supported in part by the U.S. National Science
Foundation (IOS-1239577) and by the U.S. Department of Agriculture
National Institute of Food and Agriculture (Hatch project no. CA-D*MCB-4126-H).
* Address correspondence to [email protected].
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: J. Clark Lagarias ([email protected]).
W.H. and J.C.L. conceived the project and wrote the article; W.H.
designed the research, performed experiments, and analyzed data;
J.C.L. supervised the project.
[OPEN]
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366
biotechnology will play a key role in sustaining and increasing crop yield on the limited arable land available
(Mittler and Blumwald, 2010).
As master regulators of shade avoidance (Casal, 2013),
phytochromes are obvious targets for conventional and
molecular breeding programs designed to modify plant
stature, alter the timing of flowering, and/or improve
crop harvest index at high planting densities (Robson
et al., 1996). The pioneering investigations of Harry Smith
more than 20 years ago well illustrate the agronomic
potential of phytochrome A (PHYA) overexpression to
suppress shade avoidance behavior in crop plant species
(Smith, 1994; Robson and Smith, 1997). Phytochromebased technologies have not been widely adopted since
those studies, however, possibly due to unpredictable
negative consequences of constitutive overexpression on
plant performance. In this study, we leverage dominant,
gain-of-function YHB alleles of phytochrome B (phyBY276H) placed under control of tightly regulated
promoter-operator sequences. YHB-mediated suppression of shade avoidance behavior is retained under farred (FR)-enriched shade light—conditions that inactivate
endogenous phyB (Su and Lagarias, 2007; Hu et al., 2009).
Regulated YHB expression therefore permits unprecedented control of phytochrome signaling in a manner
independent of the ambient light fluence rate and light
quality in the environment. Here, we detail research to
tailor the YHB allele as a robust genetic selection marker
in the model plant species Arabidopsis (Arabidopsis
thaliana)—an approach that is easily applicable to other
plant species. YHB’s intense red fluorescence provides the
added benefit of the ability to noninvasively monitor its
expression in vivo.
Genetic selection markers are critical components of
molecular breeding programs for crop improvement. Selectable markers using chemicals as selection agents that
confer resistance to phytotoxic substances such as antibiotics and herbicides, enable utilization of unconventional
or elevated levels of conventional metabolites, and exploit
overproduction of the phytohormone cytokinin have been
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Engineered PhyB-Based Marker Genes
well reviewed (Miki and McHugh, 2004; Rosellini, 2012).
In some cases, plant gene-derived selection markers that
use chemical selection agents have been developed
(Rosellini, 2011). The rice OsHSP101 is the best-known
marker gene exploiting abiotic physical treatment, i.e.
heat shock (HS), as the selection stress (Chang et al.,
2007). Selectable markers that leverage seed-specific
promoter to tightly express fluorescent proteins for
visual indication are steadily gaining application in
Arabidopsis (Stuitje et al., 2003; Shimada et al., 2010).
For these applications, transgenic fluorescent seeds can
be rapidly and efficiently screened under fluorescence
stereomicroscope without use of added chemicals. By
contrast, we employ a HS-inducible promoter to regulate constitutively active YHB alleles, representing a
new class of chemical-free, plant gene-based markers
for facile, low-cost transgenic screening applications.
RESULTS
Proof-of-Concept Studies
We initially examined seed germination and seedling
development of T1 populations of constitutively expressing 35S::YHB transformants of Arabidopsis (Landsberg
erecta [Ler] ecotype) grown under dim/intermittent light
or in darkness (Fig. 1). YHB-expressing transformants
could be identified by their constitutive photomorphogenic (cop) phenotype with short hypocotyls and fully
expanded cotyledons that were easily distinguished from
a taller lawn of nontransformants (Fig. 1A). On Murashige
and Skoog growth medium, nontransformant seeds
exhibited robust germination even when exposed to FR
but could easily be distinguished from YHB lines by their
etiolated phenotype. We found that nontransformant
germination could be fully inhibited by prior FR irradiation when seedlings were grown on phytagar
plates lacking Murashige and Skoog salts. Under these
conditions, YHB lines were easily distinguished from
nontransformants by their ability to germinate in
darkness after FR and also by their cop developmental
phenotype (Fig. 1B).
Removal of Murashige and Skoog salts from the
phytagar medium also proved necessary for successful
propagation of YHB transformants grown in darkness.
Following light exposure, 4-d-old dark-grown YHB
transformants photobleached and died in the presence
of Murashige and Skoog salts. By contrast, seedlings
grown on phytagar medium lacking Murashige and
Skoog salts survived—a phenotype associated with
reduced protochlorophyllide levels (Supplemental Fig.
S1, A and B). Protochlorophyllide was fully converted
into chlorophyll(ide) following 4 h light exposure for
phytagar-grown YHB transgenics, whereas excess protochlorophyllide remained in those grown on Murashige
and Skoog. Quantitative reverse transcription PCR (qRTPCR) measurements established that the light-sensitive
phenotype reflects reduced NADPH:protochlorophyllide
oxidoreductase A (PORA) expression in YHB transformants
grown on Murashige and Skoog (Supplemental Fig. S1C).
Figure 1. The pHSP::YHB selection cassette confers heat shock (HS)dependent morphological traits for transgenic selection. A and B, Screen
feasibility tests of T1 35S::YHB transformants under dim light (A) or under
true darkness on phytagar plate (B). Arrows indicate positive transformants.
C, Diagram of the pHSP01-YHB vector (T-DNA region) for dual selection
by either kanamycin or HS. Unique restriction sites are indicated. D, Fourday-old seedlings grown under various light conditions with or without
daily 2 h 37°C HS. P, Nontransformant parent; T, homozygous T3 pHSP::
YHB line; WT, wild type. E, Twenty-six-day-old plants grown under shortday conditions with or without daily 2 h 37°C HS. YHBg, genomic YHB
lines driven by the native PHYB promoter. Bars = 1 cm.
This photosensitivity therefore arises from the excess accumulation of non-PORA-bound protochlorophyllide,
which is strongly photodynamic.
To mitigate the effects of constitutively expressed
YHB on plant growth, we examined promoter-operator
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Hu and Lagarias
systems that could be selectively and/or transiently activated. The Arabidopsis HSP18.2 HS promoter has been
widely used in plant biotechnology for tight temporal
induction of target genes of interest (Takahashi et al., 1992;
Yoshida et al., 1995; Matsuhara et al., 2000; Masclaux
et al., 2004; Luo et al., 2008). In this regard, HSP18.2 expression is nearly undetectable in 4- to 7-d-old seedlings
(Hu and Ma, 2006; Hu et al., 2009) as well as in most adult
plant tissues maintained at/or below 25°C (Supplemental
Fig. S2; Tsukaya et al., 1993). Since daily 37°C treatments
up to 8 h/d did not visibly alter seedling development of
YHB transgenics compared with control YHB lines held at
22°C (Supplemental Fig. S1D), the HSP18.2 promoter was
chosen for more in-depth analyses.
For side-by-side comparison of traditional antibiotic
and HS selection efficiencies, we constructed a vector
containing both pNOS::NPTII and pHSP::YHB cassettes
(Fig. 1C). T1 seeds from multiple transformation events
were mixed and divided into two equal batches. One
was subjected to kanamycin selection while the other
was exposed to daily 2 h 37°C treatments (2 h HS).
These studies showed that the frequency of kanamycinresistant transformants was comparable with those
exhibiting a HS-induced cop phenotype (Table I), illustrating the practical utility of pHSP::YHB cassette for
genetic selection. Moreover, positive transformants
could be identified within 4 d with our HS screen, and
sometimes within 3 d. By contrast, reliable kanamycin
selection required 10 d to 2 weeks of growth.
We then compared Ler wild-type and phyB-5 mutant
parent lines with two homozygous pHSP::YHB transgenic
lines grown for 4 d in the absence or presence of daily HS.
Seedlings of all lines were fully etiolated in darkness and
were otherwise indistinguishable under continuous red
light in the absence of HS (Fig. 1D). Following daily 2 h HS
in darkness, YHB seedlings exhibited strong cop phenotypes not seen in control lines, and as expected, YHB
seedlings were hypersensitive to dim light (Fig. 1D).
Adult YHB transgenics also displayed HS-dependent
phenotypes not observed in control lines (Fig. 1E). Daily
HS treatment of pHSP::YHB/phyB lines rendered adult
plants with short petioles and compact rosettes resembling those of the well-characterized YHBg plant line
(Su and Lagarias, 2007). In the absence of HS, adult
pHSP::YHB/phyB plants could not be distinguished
from phyB mutants. These studies showed that the
Table I. Similar numbers of identified transformants indicate comparative antibiotic and heat shock selection efficiencies
Same numbers of T1 seeds (by weight) from the same seed batch of
each genotype were screened. n.a., Not analyzed.
Genotypes
Kanamycin
pHSP::YHB/Ler
pHSP::YHB/phyB-5
pHSP::eYHBN651-CNF/phyB-5
pHSP::eYHBN448-CNF/Ler
pHSP::eYHBN651-CNF/Col
pHSP::eYHBN448-CNF/Col
11
25
23
6
9
10
Heat shock
Dim Light
Dark
12
29
32
6
11
16
11
26
n.a.
n.a.
n.a.
n.a.
HSP18.2 promoter confers tight, conditional regulation
of YHB expression in transgenic plants.
Optimization of HS Induction
We next examined the duration, frequency, timing, and
temperature range of HS treatments. First tested was the
effect of HS duration on morphogenesis of pHSP::YHB
seedlings grown in darkness (Fig. 2A). Whereas daily
0.5 h HS pulses induced discernable photomorphogenesis
(not observed in controls), cop phenotypes became progressively more pronounced when the HS duration was
increased from 0.5 to 4 h. Plants given HS pulses of 4 h or
longer were phenotypically similar to YHBg seedlings
(Fig. 2A). Since selection efficiencies were identical for 2, 4,
and 6 h HS (Fig. 2A, see table), we used 2 h pulses to
optimize the timing and/or frequency of HS treatments.
These studies showed that a single 2 h HS treatment on
day 2 was as effective as daily HS for 4 d (Fig. 2B). In
parallel with temporal HS studies, we also assessed the
effect of temperature on dark-grown seedling development. We observed no discernable photomorphogenesis
for YHB lines maintained at or below 31°C and only minor phenotypic changes for YHB lines kept at 32°C to
33°C in darkness (Fig. 2C). Only when temperatures
exceeded 38°C was growth visibly inhibited, while
temperatures over 42°C strongly inhibited seed germination (Fig. 2C). Temperatures of HS pulses between 34°C and 37°C were therefore chosen as optimal
for a robust screen, consistent with earlier studies on
pHSP::GUS reporter lines in Arabidopsis and petunia
(Petunia hybrida; Takahashi et al., 1992).
To determine whether the extent of YHB expression
correlates with seedling cop phenotypes, we analyzed
YHB protein levels by immunoblotting (Fig. 3A). Undetectable in pHSP::YHB/phyB-5 seedlings prior to HS,
YHB accumulated to a level comparable with that of
endogenous phyB in the wild type by the end of the 2 h
37°C HS treatment. Moreover, YHB levels continued to
increase for the next 2 h, remaining elevated up to 24 h
but dropping significantly from 36 h to 72 h. Since YHB
is strongly red fluorescent (Su and Lagarias, 2007), we
also used confocal microscopy to detect its accumulation
and subcellular localization in dark-grown YHB lines.
Visible YHB nuclear bodies could only be detected 6 h
after HS, considerably later than maximal protein accumulation (Fig. 3B). These results are consistent with time
lag needed for bilin chromophore binding, nuclear migration, and nuclear body formation (Kircher et al., 2002;
Kevei et al., 2007). The prolonged nuclear accumulation
of YHB well illustrates the effectiveness of a single HS on
day 2 for phenotypic selection (Figs. 2B and 3).
Re-Engineering YHB Alleles
With the success of proof-of-concept studies, we next
sought to simplify our constructs to enhance the utility
of YHB as a selection marker. Since the YHB open
reading frame contains many undesirable restriction
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Engineered PhyB-Based Marker Genes
The full-length YHB coding sequence is large, and the
region encoding the C-terminal domain is dispensable
for wild-type phyB activity (Matsushita et al., 2003;
Chen et al., 2005; Park et al., 2012). We therefore truncated
the full-length YHB gene and replaced the C-terminal
domain-encoding sequence (521 amino acids) with a
much shorter one encoding CPRF4a dimerization domain
(46 amino acids; Kircher et al., 1998) fused to a nuclear
localization signal sequence (NLS) and a 3xFLAG tag
(abbreviated here as CNF). The latter substitution has
been shown to generate a PHYB chimera that retains
signaling activity (Palágyi et al., 2010). In addition to
generating the pHSP::eYHBN651-CNF construct (Fig. 4A;
Supplemental Fig. S5), the more truncated constructs
pHSP::eYHBN448-CNF and 35S::eYHBN448-CNF (Fig. 4A;
Supplemental Fig. S6) were also assembled. An early
study showed that further removal of the PHY domain
did not abolish phyB function but may shorten the
lifetime of active Pfr-phyB (Oka et al., 2004). Since YHB
is locked in the physiologically active state (Su and
Lagarias, 2007), we hypothesize that the N448 chimera
may also retain signaling activity.
Expression of the full-length and two truncated constructs in planta was next determined immunochemically. As expected, FLAG-tagged chimeric proteins of the
expected molecular sizes were observed in HS-induced
transgenic seedlings (Fig. 4B). The intrinsic red fluorescence of YHB provides a means for its detection in living
tissues by confocal microscopy (Su and Lagarias, 2007).
Such measurements documented the accumulation of
Figure 2. Optimization of HS screening protocol. A, Effect of daily HS
duration on growth in darkness, and comparison of screen efficiency
with 2, 4, and 6 h daily HS (below). B, Effects of the number and timing
of HS on growth in darkness showing that one 2 h HS on day 2 (postgermination) is sufficient. The bottom panel shows dark-grown seedling
development in the absence of HS over the 4 d period. C, Effects of HS
temperature (applied 2 h per day) on growth in darkness. Optimal induction range is 34°C ; 37°C; higher temperature suppresses growth
and/or germination. Bars = 1 cm.
sites, we ablated 25 sites by introducing synonymous
mutations. To the resulting eYHB allele, we also added
a 3xFLAG sequence (Supplemental Figs. S3 and S4A).
Transgenic lines expressing 35S::YHB or 35S::eYHBFLAG were then phenotypically compared with the
wild type. Both lines exhibited similar cop phenotypes,
confirming functional equivalence of eYHB-FLAG and
YHB alleles (Supplemental Fig. S4B). Immunoblot analyses also documented the expression of eYHB-FLAG
protein using both anti-phyB and anti-FLAG antibodies
(Supplemental Fig. S4C).
Figure 3. Induction and stability of YHB proteins following 2 h HS
(37°C) of dark-grown, 3-d-old seedlings. A, Immunoblot detection of
YHB protein levels upon 2 h HS. B, Confocal fluorescence microscopy of 49,6-diamino-phenylindole (DAPI)-stained nuclei (blue) and
fluorescent YHB (red) indicates that YHB nuclear bodies form ;6 h
following HS and remain for at least 48 h. DIC, Differential interference contrast.
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Hu and Lagarias
Figure 4. Truncation of YHB selection
cassettes. A, Domain architectures of
full-length eYHB and C-terminal regulatory domain deletion constructs fused
to the CNF peptide that encodes a dimerization domain, NLS, and 3xFLAG
epitope tag; CPRF4a-DD, the 46-residue
dimerization domain of CPRF4a (see
“Materials and Methods” for details). B,
Immunoblot validation of in planta
expressed chimeric proteins using antiFLAG antibody; values on the right and
left are the theoretical molecular masses
(kD) of chimeric peptides and molecular
mass standards, respectively. C, Red
fluorescence of both N651 and N448
chimeric proteins are evenly dispersed
in nuclei of dark-grown transgenic
seedlings, in contrast to forming large
nuclear bodies seen for full-length
eYHB; bar = 5 mm. DAPI, 49,6-Diaminophenylindole; DIC, differential interference contrast. D, Immunoblot analysis
of eYHB chimeras and PIF3 protein
levels from 4-d-old, dark-grown seedlings subjected to daily 2 h HS. E, Phenotypes of 5-d-old seedlings, and mean
lengths (6SD) of 15 hypocotyls are shown;
bar = 1 cm. F, Cotyledon area of 4-d-old
seedlings in darkness or under dim white
light given daily 2 h HS; mean area (6SD)
of 20 cotyledons are shown; bar = 1 mm.
G, HS treatment does not induce dark
germination of pHSP::eYHBN448-CNF,
but may slightly promote germination of
pHSP::eYHBN651-CNF. The details for
light and HS treatments are described in
“Materials and Methods.” Means 6 SD
are presented from at least two independent lines and $ four replicates.
YHB in the nucleus (Fig. 4C). Similar to the previous
studies (Matsushita et al., 2003; Oka et al., 2004; Chen
et al., 2005; Palágyi et al., 2010), the truncated YHB proteins were diffusely distributed in the nucleus and did
not form the discrete nuclear bodies seen for full-length
eYHB. Immunoblot analysis revealed that the protein
levels of HS-induced N651 and N448 were significantly
higher than that of full-length eYHB (Fig. 4D).
Phenotypic Selection of eYHB Transformants
Both truncated eYHB constructs generated transgenic
lines exhibiting HS-dependent cop phenotypes that
were also hypersensitive to dim light; however, their
hypocotyl length and cotyledon expansion phenotypes
were less apparent than those of full-length eYHB lines
(Fig. 4, E and F). The HS screen efficiency for both
truncated constructs proved similar to that of antibiotic
selection (Table I). pHSP::N651 transformants were
readily identified by their cop phenotype. Although also
recognizable, the smaller cotyledons of the pHSP:N448
lines made identification of transformants a bit more
subjective (Fig. 4F). In the Columbia (Col) ecotype, dim
light screens of pHSP::N448 and pHSP::N651 lines were
also feasible, with the N651 construct generally conferring the stronger cop phenotype (Supplemental Fig.
S7; Table I). The phenotypic difference between N651
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Engineered PhyB-Based Marker Genes
and N448 suggests that the PHY domain plays a
greater role in phyB signaling than merely stabilizing
the Pfr form.
Comparative immunoblot analyses revealed that
HS-induced accumulation of truncated YHB proteins
were comparable with, albeit slightly less than, that of
35S::N448 lines. By contrast, HS-induced protein levels
of full-length YHB in the eYHB lines were dramatically
lower than those in all three truncated lines (Fig. 4D).
qRT-PCR measurement showed that HS-induced eYHB
transcript levels were similar to or 4-fold less than those
of truncated eYHB variants (Supplemental Fig. S8). This
suggests that translational and/or posttranslational
regulation rather than transcriptional regulation mainly
account for the low protein levels of full-length YHB. For
each genotype, however, we observed a good correlation
between transcript and protein levels of the two transgenic lines (Fig. 4D; Supplemental Fig. S8). The elevated
N448 levels in the 35S::N448 lines did not enhance their
cop phenotypes in comparison to HS-treated pHSP::N448
lines, showing that HS-induced N448 protein levels are
sufficient for saturating their signaling activity (Fig. 4, D–
F). 35S::N448 partially complemented the phyB-5 mutant
under continuous red light; both 35S::N448 and pHSP::N448
failed to promote light-independent seed germination
(Fig. 4G; Supplemental Fig. S9). The N651 construct promoted a low level of seed germination (Fig. 4G). These
results indicate that truncated YHBs are less active than
the full-length YHB, consistent with a recent study using
the same CPRF4a dimerization domain (Palágyi et al.,
2010) but contrary to the earliest finding that phyBN651GFP-GUS-NLS was more active than phyB-GFP
(Matsushita et al., 2003).
As another measure of YHB activity, we next determined the level of PHYTOCHROME INTERACTING
FACTOR3 (PIF3) protein, one of the key transcription
factors targeted for degradation by light-activated phyB
(Ni et al., 2014). These immunoblot analyses indicate that
YHB promoted degradation of PIF3 in darkness—a hallmark of light signaling by phyB (Fig. 4D). By contrast,
N651 did not significantly reduce the levels of PIF3, even
though it strongly triggered photomorphogenesis in the
dark. This result is consistent with a recent study showing
that the N-terminal photosensory region of phyB (N651)
can inactivate PIFs but does not trigger PIF degradation
(Park et al., 2012). However, PIF3 levels were unexpectedly reduced in N448 lines in both Ler and Col ecotypes
(Fig. 4D; Supplemental Fig. S7G). While the explanation
for this result is currently unclear, our findings show that
the phenotypic consequences of full-length and truncated
YHB expression do not strictly correlate with the observed PIF3 protein levels.
eYHBs Are Sensitive Monitors of Off-Target Expression
While we obtained pHSP::(e)YHB lines that do not express YHB at nonpermissive temperatures, roughly 80%
of pHSP::(e)YHB lines exhibited “leaky” misexpression as
revealed by their cop development in darkness in the
absence of HS (Fig. 5A). These HS-independent cop phenotypes can be classified as strong (comparable to their cop
phenotypes after HS), moderate (having elongated hypocotyls with enlarged cotyledons), or weak (with long
hypocotyls and partially opened cotyledons). Such leakiness could reflect read-through transcription from the
adjacent NOS promoter that drives the NPTII marker gene
(see pHSP01 constructs in Fig. 5B), and/or the influence of
T-DNA and/or plant enhancer sequences flanking the
inserted transgene. In this regard, enhancers within the
T-DNA have been reported to override tissue- and/or
Figure 5. Leaky misexpression of the pHSP::YHB cassettes. A, Representative leaky cop phenotype of pHSP::eYHB seedlings in the absence of heat treatment (two independent lines present each
phenotypic class); bar = 1cm. B, Distribution of leaky cop phenotype
of transgenics obtained from three different selection constructs as
shown. One hundred and seven, 97, and 56 independent lines were
analyzed for pHSP01-eYHB, pHSP03-eYHB, and pHSP01-N651
constructs, respectively.
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Hu and Lagarias
development-specific expression pattern of cotransforming promoter sequences (Yoo et al., 2005; Zheng
et al., 2007).
To distinguish between these two possible explanations for (e)YHB misexpression, we designed and tested
a pHSP03-eYHB transformation construct that lacks the
entire pNOS::NPTII::tNOS selection cassette (Fig. 5B).
Selection experiments revealed this construct to increase
the percentage of nonleaky lines from 18% to 40%, while
also decreasing the percentage of strong leaky lines from
50% to 20% (Fig. 5B). Polar effects of enhancers near the
site of eYHB insertion are therefore likely responsible for
the remaining misexpression of pHSP03-eYHB in the
absence of HS. Interestingly, substitution of full-length
eYHB with the truncated N651 increased the recovery of
nonleaky lines to 84% irrespective of the presence of the
pNOS::NPTII::tNOS selection cassette within the same
construct (Fig. 5B). Similar results were observed in the
Col background transformed with pHSP01-N651 (10%
lines marginally leaky) and with pHSP01-N448 (no leaky
line found; Supplemental Fig. S7F). As such, pHSP::(e)
YHB cassettes therefore are valuable tools for identifying
transformants in which the targeted gene of interest is
inserted at a neutral locus and retains tight regulation by
the promoter-operator sequence as designed. On the
other hand, both truncated YHB selection cassettes tolerate the leaky misexpression owing to their weaker
signaling activities that require a high expression level to
confer discernable cop phenotypes; they can be safely
used in traditional transgenics employing strong viral promoters such as 35S and NOS to drive overexpression of the gene of interest.
DISCUSSION
In this report, we validate a novel dominant plant
gene-based selectable marker cassette consisting of a
HS-inducible promoter for tight regulation of a phyB
allele with a single missense mutation (YHB). Genes
encoding both components are already present ubiquitously in plant genomes, including those of crop species. The introduced single missense mutant variants of
phyB are thus unlikely to trigger immune responses or
other unexpected consequences if/when consumed by
humans or livestock. Our selection system circumvents
the need for antibiotics or herbicides for identification of
transformants that are easily distinguished from untransformed plant lines in darkness or under low light
without the need for specialized or expensive equipment. We anticipate that this approach can be generalized by replacing YHB with other constitutively active
photoreceptors, including Arabidopsis cryptochromes
CRY1G380R (Gu et al., 2012), CRY1W400A (Gao et al.,
2015), and CRY2W374A (Li et al., 2011), and UV-B photoreceptor UVR8W285A (Heijde et al., 2013). In this manner,
conditional expression of constitutively active photoreceptors should prove a powerful approach for probing
their light signaling pathways in a manner independent of
the light environment.
YHB-based selectable markers should be easily applicable to crop plants that are in planta transformable
to directly produce transgenic seeds and have distinct
dark- and light-grown seedling morphology. An increasing number of vegetable and oilseed crops in the
Brassicaceae family can be transformed by floral dip
plus vacuum infiltration, such as Brassica napus (Wang
et al., 2003; Li et al., 2010; Tan et al., 2011), Brassica rapa
(Xu et al., 2008; Mao et al., 2014), Brassica juncea (Dai
et al., 2011), and radish (Raphanus sativus; Curtis and
Nam, 2001). These crucifer crops are usually grown in
cool seasons and less likely exposed to temperature
higher than 34°C to trigger pHSP::YHB transcription to
impact plant growth. Whereas Brassica plants that
flower and set seed in late spring may experience a
short period of high temperature, the resulting expression of YHB might even enhance photosynthesis
and delay senescence potentially leading to an overall
benefit to crop yield. Successful in planta transformations were also reported from other eudicot and
monocot plants, including Medicago truncatula (Trieu
et al., 2000), soybean (Glycine max; Gao et al., 2007),
tomato (Lycopersicon esculentum; Yasmeen et al., 2009),
wheat (Zale et al., 2009), maize (Zea mays; Mu et al.,
2012; Moiseeva et al., 2014), and Setaria viridis (Martins
et al., 2015).
Most plant species require selection in tissue culture
prior to regeneration of transformant lines. In these
cases, cotransformation can be performed with a second plasmid or a second T-DNA unit containing an
antibiotic selection marker for selection in tissue culture
(Daley et al., 1998; Matthews et al., 2001; Matheka et al.,
2013). Following regeneration, the antibiotic resistance
marker can then be removed by segregation in subsequent sexual generations. Afterward, the YHB marker
and its linked gene of interest allow facile phenotypic
identification of transgenic lines, thus alleviating molecular assay workload for this purpose. Due to its
bright red fluorescence and nuclear location, eYHBexpressing transformants potentially can be identified
in tissue culture by wide-field fluorescence microscopy.
It is also conceivable that YHB transformants will exhibit altered hormone sensitivity, enabling development of selective regeneration protocols that can be
used for direct selection in crop plant species.
The utility of eYHB alleles for monitoring off-target
expression of inserted transgenes illustrates another
advantage of our approach. Even when expressed at
low levels, eYHB transformants are easily identified
among dark-grown seedlings. As such, eYHB alleles are
sensitive indicators of misexpression due to cis-acting
flanking sequences located within the T-DNA construct
itself and/or those present in the plant genome near the
site of transgene insertion. Our analyses show that transgene misexpression occurs at a much higher frequency
than expected. Even the NOS promoter—previously
deemed a “safe” replacement for the enhancer-rich 35S
promoter (Zheng et al., 2007)—was able to override
the tight HS dependence of the pHSP promoter. The
pHSP::eYHB cassette thereby represents a new tool to
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Engineered PhyB-Based Marker Genes
assuage concern that the transgene of interest is only
expressed in a desired spatiotemporal pattern. By contrast, off-target expression of GFP and GUS reporters is
easily overlooked by subjective adjustments in exposure
times used for visualization of expressed transgenes.
Genetic complementation also does not ensure that the
observed GFP/GUS expression pattern truly represents the
biologically significant fraction of the expressed protein. In
conjunction with a desired gene of interest, eYHB alleles
should be useful for identifying “neutral sites” that do not
interfere with the specificity of the regulatory sequences
used to control the expression of the transgene. Indeed, a
more systematic analysis of regulatory sequences is needed
to secure even more tightly regulated promoter-operator
combinations critical for the success of synthetic biology
applications (Liu and Stewart, 2015; Ghareeb et al., 2016).
Genetic selection of plants has played a pivotal role in
the development of nutritional, high-producing varieties of food crop species that have so far sustained the
growth in human population. As human activity dramatically impacts the climate of our planet, particularly
in the midlatitudes, rapid development of new varieties
of stress-tolerant varieties of crop plant species will be
essential. While phytochromes play a major role in
entraining plant metabolism to diurnal changes in light
fluence rate and seasonal changes in day length, their role
as temperature sensors is becoming increasingly more
appreciated (Franklin et al., 2014; Quint et al., 2016). It is
well established that light quality and light fluence rate
both influence the signaling activity of phyB due to a
process known as dark reversion. Dark reversion is a
temperature-dependent process in which the signalingactive Pfr form of phyB nonphotochemically reverts to
the inactive Pr state. This process is accelerated under low
fluence rates of light and under FR-enriched shade light
where most of the signaling-active Pfr form is present as a
heterodimer with an inactive Pr subunit (Brockmann
et al., 1987). Dark reversion is strongly implicated in the
temperature-sensing role of phytochromes, e.g. temperatureenhanced floral induction (for review, see Quint et al.,
2016). Since YHB does not dark revert (Su and Lagarias,
2007), its expression hold great potential for suppression
of the temperature-dependent inactivation of endogenous phyB to confer enhanced thermotolerance of crop
plant species on a warming planet. Indeed, a recent
study indicated that constitutive YHB expression greatly
suppresses high temperature-induced transcriptomic
and morphological changes in Arabidopsis (Jung et al.,
2016). Temperature-regulated expression of YHB thus
holds great potential for crop plant species such as alfalfa (Medicago sativa) where early flowering in response
to HS is deleterious to forage quality.
generate the pHSP01 construct used in this study. pHSP01 was digested with KpnI
and XbaI and ligated with the full-length Arabidopsis (Arabidopsis thaliana) PHYBY276H (YHB) sequence similarly excised from pJM61-35S::YHB (Su and Lagarias,
2007) to generate pHSP01-YHB. For the designed eYHB-3xFLAG sequence
(Supplemental Fig. S3), codons frequently found from the endogenous PHYB open
reading frame sequence were chosen for generation of synonymous mutation of
25 targeted restriction sites. A HindIII site in the CPRF4a dimerization domain
(Kircher et al., 1998; Palágyi et al., 2010) was similarly mutated. eYHB-3xFLAG and
CPRF4a-NLS-3xFLAG (abbreviated as CNF) sequences were synthesized by
GenScript. Truncated eYHBN651 and eYHBN448 fragments were amplified using
eYHB-3xFLAG as template and cloned into KpnI and XhoI sites of pBluescript. CNF
was ligated with pBS-eYHBN651 and pBS-eYHBN448 via XhoI and XbaI sites. eYHBFLAG, eYHBN651-CNF, and eYHBN448-CNF (Supplemental Figs. S5 and S6) were
cloned into pHSP01 via KpnI and XbaI ligation. eYHB-FLAG and eYHBN448-CNF
were also cloned into pJM61 as 35S-driven overexpression constructs. pHSP03eYHB was created from pHSP01-eYHB by replacing the pNOS::NPTII::tNOS
selection cassette with a linker duplex (PmeI-SalI-BamHI-PacI-StuI-SpeI-ApaI-ClaI) via
PmeI and ClaI ligation. Oligonucleotides used for plasmid construction and subcloning are listed in Supplemental Table S1. All constructs were transformed into
wild-type Arabidopsis (Ler or Col-0 accessions) and the phyB-5 mutant by the floral
dip method (Clough and Bent, 1998) to make transgenic plants.
Standard HS Screening Protocol
Plant seeds were surface sterilized with 70% ethanol for 15 min, then
resuspended in 0.1% phytagar solution and sown on half-strength Murashige
and Skoog growth medium (pH 5.7, 0.75% phytagar, no Suc). After 4 d imbibition
in the cold room, plates were exposed to white light for 3 h to synchronize seed
germination. A standard dim light HS screen for positive transformants and
phenotypic analyses were performed in a Conviron growth chamber at 20°C
under 8 h light (0.3 ; 1.5 mmol m22 s21)/16 h dark cycles. Daily 2 h 37°C HS
treatments were administered at subjective noon. Alternatively, a microbiological incubator set at 34°C to 37°C was used for low-cost HS screen; plates
were kept in the dark except on day 2 and day 3 when they were given a brief
light exposure (;10 mmol m22 s21 for 30 to 60 min) to prevent photodamage for
YHB-expressing seedlings otherwise grown in constant darkness through day
4. For HS screen under true darkness, seeds were sown on 0.8% phytagar plates,
imbibed as above, exposed to FR (12 mmol m22 s21) for 5 min, kept in darkness,
and subjected to daily 2 h HS treatments. YHB seedlings grown in darkness in
the absence of Murashige and Skoog salts can green after switch to light growth
without a brief low light treatment.
Seed Germination Assay
Seed germination assay essentially followed a previous method (Oh et al.,
2004). Approximately 100 seeds of each genotype were surface sterilized, sown
on 0.75% phytagar plate (pH 5.7), and within an hour exposed to FR (12 mmol
m22 s21) for 5 min. Some were then allowed to germinate in white light for 5 d,
exposed to red light (20 mmol m22 s21) for 5 min followed by 5 d dark growth, or
kept in darkness for 5 d with or without HS (single or daily repeated treatments). Seeds were deemed germinated beyond radicle emergence.
Protein Extraction and Immunoblot Assays
Protein extraction and immunoblot analysis were performed as described previously with some modifications (Su and Lagarias, 2007; Jones et al., 2015). Immunoblot visualization of FLAG-tagged proteins, actin, and tubulin were accomplished
using the fluorescence-based Odyssey infrared imaging system as described previously, and PIF3 and YHB/PHYB proteins were detected by the ECL method using
the GE ImageQuant LAS4000 system. Representative immunoblot results from at
least two replicates are presented. Blot intensities were quantified using LI-COR
Image Studio software (for FLAG and actin) or using NIH ImageJ (for PIF3).
qRT-PCR
MATERIALS AND METHODS
Plasmid Constructs and Plant Transformation
The pTT101 plasmid (Matsuhara et al., 2000) obtained from Arabidopsis Biological
Research Center was modified to introduce a unique KpnI site by digestion with SmaI
and XbaI and religation with a short duplex linker (SmaI-KpnI-BamHI-SpeI-XbaI) to
Dark-grown, 4-d-old seedlings subjected to daily 2 h HS were harvested
under dim green light 2 h after the last HS and immediately frozen in liquid
nitrogen. The two 35S::eYHBN448-CNF/phyB-5 lines grown without HS treatment were also harvested at the same time. Total RNAs were extracted using
RNeasy Plant Mini Kit (Qiagen) following manufacturer’s instructions. One microgram of DNase I-treated RNA was used for cDNA synthesis using iScript
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Hu and Lagarias
Reverse Transcription Supermix (Bio-Rad). Quantitative PCRs were performed using the SsoAdvanced Universal SYBR Green Supermix in the
CFX96 real-time PCR detection system (Bio-Rad). Primers for detecting
transcript levels of endogenous PHYB and all YHB variants were 59GTGCTGTTCAATCGCAGAAA-39 and 59-TCCACGACAGTGTCACACAA-39.
Primers for HSP18.2 were 59-AAGGCAACAATGGAGAATGG-39 and 59TATTCAATTAGCCCCGGAGA-39. Primers for the reference gene UBQ10
(At4g05320) were the same as before (Hu et al., 2009).
Miscellaneous
Confocal fluorescence microscopy was performed as described previously
(Su and Lagarias, 2007); more than three seedlings were examined for each
genotype or growth condition. Light fluence rates were measured with a BlackComet-CXR-SR spectrometer (StellarNet) or an LI-189 photometer (LI-COR).
Hypocotyls and cotyledons of seedlings were quantified by analyzing photos
with NIH ImageJ.
Accession Numbers
Sequence data of the loci investigated in this study can be found in the
Arabidopsis Information Resource under the following accession numbers:
At2g18790 (PHYB), At5g59720 (HSP18.2), and At1g09530 (PIF3).
Supplemental Data
The following supplemental data are available online.
Supplemental Figure S1. Additional feasibility tests for YHB as a selectable marker gene.
Supplemental Figure S2. Expression estimates of HSP18.2 decoded from
Affymetrix microarray datasets, and its comparison to that of PHYB.
Supplemental Figure S3. Synthesized eYHB-3xFLAG sequence.
Supplemental Figure S4. Engineered YHB (eYHB-FLAG) and functional
validation.
Supplemental Figure S5. Annotated eYHBN651-CNF sequence.
Supplemental Figure S6. Annotated eYHBN448-CNF sequence.
Supplemental Figure S7. Application of pHSP::eYHBN651-CNF (abbreviated as N651) and pHSP::eYHBN448-CNF (N448) selectable cassettes in
the Col wild type.
Supplemental Figure S8. qRT-PCR examination of HS-induced YHB and
truncated YHB transcript levels.
Supplemental Figure S9. eYHBN448-CNF in the phyB mutant is weakly
functional in darkness but can mostly complement seedling growth of
phyB mutants under continuous red light.
Supplemental Table S1. Oligonucleotides used for construct engineering
and subcloning.
ACKNOWLEDGMENTS
We thank Drs. Eva Adam and Ferenc Nagy for details involving the CPRF4a
dimerization domain; Drs. Peter Quail and Meng Chen for generous gifts of
PHYB and PIF3 antibodies, respectively; Iniyan Ganesan for assistance in
phenotypic analysis; and Drs. Savithramma Dinesh-Kumar and Dazhong
Zhao for critical reading of the manuscript.
Received August 25, 2016; accepted November 22, 2016; published November
23, 2016.
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