Plant Physiol. (1999) 119: 1497-1506
Overexpression of Arabidopsis Phytochrome B Inhibits Phytochrome
A Function in the Presence of Sucrose1
Timothy W. Short*
Biology Department, Queens College and the Graduate School of The
City University of New York, Flushing, New York 11367
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ABSTRACT |
Overexpression of phytochrome B
(phyB) in Arabidopsis has previously been demonstrated to result in
dominant negative interference of phytochrome A (phyA)-mediated
hypocotyl growth inhibition in far-red (FR) light. This phenomenon has
been examined further in this study and has been found to be dependent
on the FR fluence rate and on the availability of metabolizable sugars
in the growth medium. Poorly metabolized sugars capable of activating
the putative hexokinase sensory function were not effective in
eliciting the phytochrome interference response. Overexpressed phyB
lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR.
Overexpressed phyB produces the dominant negative phenotype without any
apparent effect on phyA abundance or degradation. It is possible that
phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.
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INTRODUCTION |
Light is among the most important environmental factors
influencing plant growth and development. In the dark Arabidopsis seedlings exhibit a typical etiolated growth habit, with an elongated hypocotyl, an apical hook, and small, pale, closed cotyledons. Seedlings grown under white light progress through photomorphogenic development, which results in decreased hypocotyl-elongation rates, no
apical hook, and expanded green cotyledons. The developmental changes
are reflected in altered patterns of gene expression, cellular
activities, and biochemical properties. These responses are mediated
through a variety of photoreceptors including the five-member
phytochrome family, at least two blue/UV-A light receptors, and one or
more UV-B photoreceptors.
The Arabidopsis phytochromes, a family of homologous proteins
designated phyA, phyB, phyC, phyD, and phyE (Sharrock and Quail, 1989
;
Clack et al., 1994), absorb primarily in the red and FR regions of the
spectrum. The functions of each member of this gene family have not yet
been fully determined, but analysis of mutation and overexpression of
different phytochromes indicates that there are both overlapping and
distinct processes controlled by each gene product (Boylan and Quail,
1989, 1991; Nagatani et al., 1991, 1993; Somers et al., 1991; Wagner et
al., 1991; Parks and Quail, 1993; Reed et al., 1993, 1994; Whitelam and
Harberd, 1994; Quail et al., 1995; Aukerman et al., 1997).
Type I phytochrome, now known to consist primarily of phyA in
Arabidopsis (Hirschfeld et al., 1998), is by far the most
abundant phytochrome in etiolated tissues and is produced in
the Pr form in vivo (Mancinelli, 1994). Upon phototransformation to the
Pfr form, phyA becomes degraded rapidly via a ubiquitin-mediated
process (Clough and Vierstra, 1997). This instability results in a very low abundance of phyA during extended growth in normal light
conditions. However, under FR conditions the photoconversion to the Pfr
form is considerably less efficient, and the photoequilibrium between the Pr and Pfr forms strongly favors the more stable Pr form of phyA
(Mancinelli, 1994). The remaining phytochromes of Arabidopsis do not
undergo rapid degradation in the Pr form, although there is recent
evidence that some light regulation of other phytochromes, especially
phyC, occurs in vivo (Hirschfeld et al., 1998).
The phytochrome holoproteins consist of an apoprotein of roughly 125 kD, with a linear tetrapyrrole chromophore (phytochromobilin) covalently attached to a specific Cys in the
NH2-terminal domain (Quail, 1991; Furuya, 1993;
Furuya and Song, 1994). In vitro, and probably in vivo as well,
phytochrome is active as a homodimer (Cherry and Vierstra, 1994), but
it is not yet clear whether heterodimers form (and, if so, whether they
are active) among different members of the phytochrome family in vivo.
One or more dimerization domains are thought to reside in the
COOH-terminal domain (Edgerton and Jones, 1992; Cherry et al., 1993;
Wagner et al., 1996b). A defined region of the COOH-terminal half of
phyA and phyB has also been implicated as critical for the signaling
function of the molecules (Quail et al., 1995, 1996; Wagner and Quail,
1995; Wagner et al., 1996a).
Analysis of phyA and phyB mutants has indicated
that the so-called FR high-irradiance responses in seedlings are
primarily mediated through phyA (Dehesh et al., 1993; Nagatani et al.,
1993; Parks and Quail, 1993; Reed et al., 1994; Xu et al., 1995),
whereas the majority of red high-irradiance responses and the so-called shade-avoidance response are regulated primarily through phyB (Somers
et al., 1991; Dehesh et al., 1993; Reed et al., 1993; Smith and
Whitelam, 1997). Recent discovery and analysis of null phyD
mutants
the closest homolog to the PHYB genealso indicate that phyD is active and performs specific regulatory functions in
Arabidopsis that only partially duplicate those of phyB (Aukerman et
al., 1997). Careful analysis of hypocotyl elongation in seedlings of
the wild type and of phytochrome mutants indicate no detectable effect
of phyB or phyD under continuous FR light, whereas phyA has little or
no effect on elongation under continuous red light (Dehesh et al.,
1993; Parks and Quail, 1993; Reed et al., 1993; Quail et al., 1995,
1996).
These analyses are complicated by data showing that accumulation of
some phytochromes is regulated at least in part by the levels of other
members of the phytochrome family (Hirschfeld et al., 1998). Studies of
mutants deficient in one or more phytochromes have been complemented by
the development of transgenic Arabidopsis plants expressing complete or
partial phytochrome gene products (Boylan and Quail, 1991; Wagner et
al., 1991, 1996a, 1996b; Boylan et al., 1994; Qin et al., 1997).
Transgenic expression of certain Arabidopsis phyA and phyB fragments,
especially those with products lacking the COOH-terminal region of the
molecule, have shown dominant negative effects on primarily
phyA-mediated hypocotyl elongation (Boylan et al., 1994; Wagner et al.,
1996b). However, others studying the same or similar transgenic plants
did not report this dominant negative response in the
phyB-overexpressing lines (McCormac et al., 1993).
Physiological and molecular studies have begun to yield information
about the interaction between light cues and their modulation by other
environmental stimuli (Cheng et al., 1992; Mohr, 1994; Short and
Briggs, 1994). Carbohydrates have long been recognized as important
regulators of growth, development, and gene expression in a variety of
systems (for review, see Madore and Lucas, 1995; Pontis et al., 1995;
Koch, 1996). Among these functions, carbohydrates have been found to
alter responsiveness to light, particularly with respect to specific
gene expression (Tsukaya et al., 1991; Cheng et al., 1992; Harter et
al., 1993; Dijkwel et al., 1996, 1997). Recent data have shown that
metabolizable sugars can overcome the phyA-specific repression of
protochlorophyllide oxidoreductases (Barnes et al., 1996; T.W. Short,
unpublished data), that Suc represses light-inducible plastocyanin
production (Dijkwel et al., 1996), and that Suc can specifically affect
seedling growth in FR light (Whitelam et al., 1993; Dijkwel et al.,
1997). However, it is unclear how carbohydrates and phyA-signaling
mechanisms interact to regulate these functions. A series of mutants in
which Suc and light responses are uncoupled (sun mutants)
have been isolated and characterized (Dijkwel et al., 1997), and
analysis of several of them suggests that gene-expression responses to Suc and light follow separate, but linked, signaling pathways.
Results from several laboratories suggest that carbohydrate status may
be sensed through metabolic modification of sugars. In particular,
sugar phosphates (Sadka et al., 1994) may regulate gene expression. The
primary sensors and transducers for this mechanism are likely the
Arabidopsis hexokinases (Jang et al., 1997). Although this transduction
system may act on expression of many sugar-responsive genes, it remains
uncertain whether the hexokinase-signaling pathway is the major
mechanism involved in carbohydrate modulation of light responses. Data
from the sun mutants support the likelihood of multiple
signaling pathways (Dijkwel et al., 1997). Whereas the sun6
mutation decreased the capacity of FR-light-grown seedlings to green
after transfer to white light in the presence or absence of Suc, the
sun7 mutation had the opposite effect. Furthermore, sun7
alters the Suc effect on cotyledon opening and hypocotyl elongation,
whereas sun6 does not appear to modulate these responses.
In this study we used Arabidopsis overexpressing a PHYB
transgene to examine further the role of sugars in modulating
phytochrome signaling. We have addressed the seemingly discrepant
reports of phyB-overexpression studies and the dominant negative effect on growth, and we have investigated possible mechanisms of Suc/light interactions and placed them in the context of previous studies.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
The transgenic ABO lines were originally produced in the No-O
ecotypic background (Wagner et al., 1991) and were introgressed three
times into ecotype RLD. Mutant phyB-expressing lines with chromophore-binding Cys-357 converted to Ser (Cys-357-Ser) were also
constructed in the No-O ecotype, and phyA-101 mutants were isolated from the RLD ecotype. The original ABO(RLD) and
Cys-357-Ser(No-O)-overexpressing transgenics and phyA-101
mutants were generously provided by Drs. D. Wagner and P. Quail.
Seeds were surface-sterilized for 20 min in 20% commercial bleach
(final sodium hypochlorite concentration, 1.05% [w/v]) and 0.01%
SDS, and then rinsed three times in sterile water. Seeds were sown on
100-mm Petri plates with sterile medium containing 0.8% (w/v) agar, MS
nutrient salts (Murashige and Skoog, 1962), and 2% (w/v) Suc, unless
otherwise noted. Plates were kept in the dark at 4°C for 2 to 7 d and were then induced to germinate by exposure to white fluorescent
light for 3 h before placement in the dark at 23°C ± 1°C
for 21 h. In experiments testing the effects of different sugars
and analogs, sterilized seeds were instead plated onto 50-mm circles of
fine nylon mesh and floated on a liquid MS salt solution, and the
sugars were added to the medium at the same time the plates were
transferred to FR light. Except where noted, seedlings were allowed to
grow under continuous FR light (8.7 µmol m
2
s1) for 72 h before they were measured. FR light was
obtained from a high-output, 735-nm LED source (Q-Beam 2001, Quantum
Devices, Barneveld, WI). Where indicated, light was filtered through
plexiglass (FRF700, Westlake Plastics, Lenni Mills, PA) to eliminate
minor red-light emissions from the LED source. Light intensity and
spectral output were measured with a spectroradiometer (LI-1800,
Li-Cor, Lincoln, NE).
Analytical Measurements
Following light treatments, seedlings were laid flat on 1% agar
plates and photographed on slide film beside a reference ruler. Slides
were projected onto a digitizing tablet (model 1212, Kurta, Banska
Bystrica, Slovakia), and hypocotyls, petioles, and roots were measured
directly using SigmaScan software (Jandel Scientific, San Rafael, CA).
Unless otherwise indicated, growth data represent results from two or
three independent replicates containing 30 to 100 seedlings per
treatment. Within each experiment the total number of seedlings was
approximately equivalent for each treatment, and all of the seedlings
were measured to avoid potential selection bias.
For western blotting, seeds were sterilized and grown as indicated
above, except they were kept for 3 d in darkness and then exposed
to continuous FR or red light before harvesting at the times indicated.
The phytochrome extractions were performed by grinding 1.5 g of
frozen tissue for 3 min in 1.5 mL of extraction buffer (100 mM Tris, pH 8.3, 140 mM
[NH4]2SO4,
50% ethylene glycol, 10 mM Na2EDTA,
2 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 2 mM phenylmethylsulfonyl fluoride, 18 mM
iodoacetamide, and 12 mM sodium metabisulfate) and
centrifuging at 25,000g for 15 min at 4°C. Supernatants
were brought to 36% (NH4)2SO4 with
a cold, saturated solution of
(NH4)2SO4
and centrifuged at 25,000g for 20 min at 4°C. Pellets were
resuspended in 100 µL of resuspension buffer (50 mM Tris, pH 7.8, 5.0 mM
EDTA, 25% ethylene glycol, and 10 mM
iodoacetamide). Two 5-µL samples were removed for protein assay by a
modified Lowry assay (Peterson, 1977), and the remainder was combined
with 20 µL of sample buffer (350 mM Tris, pH
6.8, 20% SDS, 4 mM 
-mercaptoethanol, 30% [w/v]
glycerol, and 15 mg/mL bromphenol blue). SDS-PAGE and immunoblotting on
nitrocellulose were carried out according to methods described
previously (Xu et al., 1995). Following staining with 1 mg/mL Ponceau S
in 1% acetic acid to confirm equivalent protein loads, visualization of blots was performed with the PHYA apoprotein-specific monoclonal antibody 073D (developed by Dr. J. Shanklin and generously provided by
Dr. P. Quail) and an antibody-detection system (Vectastain ABC-AP,
Vector Laboratories, Burlingame, CA) using a colorimetric substrate as
previously described (Short et al., 1992).
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RESULTS |
Overexpression of phyB and Suc Alter Growth in FR Light
To address seemingly contradictory data on the effects of
constitutive high expression of phyB, we explored the response of ABO,
phyA, and the parental wild types under a variety of
conditions. Examination of the hypocotyl-elongation response indicated
that mutations in phyA result in a loss of normal responsivity to FR light regardless of the presence of 2% Suc in the growth medium (Fig.
1), as has been indicated previously
(Nagatani et al., 1993; Parks and Quail, 1993; Reed et al., 1994;
Whitelam and Harberd, 1994). Similarly, Suc had a relatively small
effect on the growth of wild-type seedlings grown in darkness (Fig. 1A)
or in 8.7 µmol m2 s1
FR light (Fig. 1B), although the light-grown seedlings did have a
slightly enhanced growth response with added Suc. ABO yielded markedly
different rates of growth in FR light depending on the presence of
exogenous Suc. Suc resulted in growth approximately double that of ABO
seedlings without Suc or that of wild-type seedlings on Suc-containing
medium. Wild-type, phyA, and ABO seedlings in all cases
exhibited similar levels of growth after 3 d in darkness with or
without Suc.
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| Figure 1.
Light-regulated hypocotyl elongation in ABO
seedlings is dependent on Suc. Seedlings from wild type (RLD and No-O
ecotypes), phyA(RLD) mutants, and phyB(No-O)
overexpressers introgressed three times into RLD were grown as
described in ``Materials and Methods'' with (white bars) or without
(black bars) 2% Suc either in darkness (A) or in 35 µmol
m2 s1 FR light (B). No-O is included to
control for possible residual effects of the original phyB ecotype
after introgression. Error bars represent ±1 SD of 75 to
120 seedlings from replicate experiments.
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The effects of Suc and FR light on differential growth rates in
wild-type and ABO plants were maintained throughout the period of 48 to
96 h following transfer to light (Fig.
2), with relative hypocotyl-length ratios
remaining almost identical throughout the period under examination. The
differential growth rate was also maintained over a range of FR fluence
rates (Fig. 3). Within the fluence rate
window investigated, the accelerated growth in ABO plants was only
observed in the presence of Suc. These disparate rates of elongation
resulted in a 58% increase in growth over wild type at 4 µmol
m2 s1 to a 130%
increase at 35 µmol m2
s1.
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| Figure 2.
Dominant negative interference by overexpressed
phyB affects growth throughout early seedling development. Hypocotyl
elongation on 2% Suc medium was measured 2, 3, or 4 d following
transfer of the seedlings to 8.7 µmol m2
s1 FR light. Growth of original parental (No-O) and
introgressed parental (RLD) wild types are compared with ABO in two
independent experiments representing approximately 100 seedlings per
treatment. Error bars represent ±1 SE.
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| Figure 3.
Effect of FR fluence rate on dominant negative
interference. Hypocotyl elongation of ABO (black bars) and wild-type
(RLD, white bars) plants was measured after growth for 3 d at the
indicated fluence rate of continuous FR light. Results are from plants
grown on medium without (A) or with (B) 2% Suc. Error bars represent
±1 SE of 100 to 150 seedlings from independent replicate
experiments. The inset in A shows the light output from the filtered
LED sources on a logarithmic scale.
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Effects of Metabolically Active Sugars and Inactive
Analogs
To study possible mechanisms of Suc modulation of phytochrome
interactions, we examined further the role of Suc and other sugars in
mediating dominant negative phyB suppression of phyA-mediated growth
inhibition. Wild-type and ABO seedlings were grown on a series of
MS-agar plates containing different Suc concentrations. Although small
increases in growth were observed in wild-type RLD as the concentration
of exogenous Suc was increased (Fig. 4),
a much greater growth response was seen in the corresponding ABO
seedlings. Transgenic and wild-type lines were significantly inhibited
at Suc concentrations of 4% and above. At 6% Suc germination was
inhibited, and the few seedlings that did germinate grew very slowly
and died before producing primary leaves (data not shown).
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| Figure 4.
The dominant negative growth response is dependent
on Suc concentration. ABO(RLD) and RLD seedlings were placed on plates
with MS salts supplemented with the indicated concentrations of Suc.
Standard growth conditions of 1 d in darkness followed by 3 d
in FR light at 8.7 µmol m2 s1 FR light
were used, and hypocotyls were measured as described in the text. Each
measurement is the result of three independent experiments with 40 to
75 seedlings per treatment per experiment. Error bars represent ±1
SE.
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Because of the apparent existence of more than one sugar-signaling
pathway in higher plants and because of the possibility that Suc acts
through osmotic effects, we examined the influences of alternate sugars
on mediating the dominant negative phytochrome response. The results of
growth with sugars known to be transported and easily utilized in plant
cells (Suc and Glc), sugars that are osmotically active but
nonmetabolizable (sorbitol and mannitol), and sugars that can be
phosphorylated and activate the hexokinase-based sensory system but
cannot be readily metabolized (Man and 2-dG; Jang et al., 1997) are
shown in Figure 5. The disaccharide Suc and the monosaccharide Glc gave essentially indistinguishable results
in these experiments, although in other experiments a slightly lower
effectiveness of Glc was noted (not shown). Neither sorbitol nor
mannitol was able to elicit the strong dominant negative response in
ABO at the concentrations expected to provide comparable osmotic
conditions. Man at 2% and 2-dG at 0.02%concentrations sufficient to
activate the hexokinase sensory/regulatory systempartially inhibited
growth and eliminated the differential between ABO and RLD wild type.
Additionally, Fru, maltose, and raffinose, but not Gal, were nearly as
effective as Suc and Glc at eliciting the ABO response (data not
shown).
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| Figure 5.
Effects of different sugars on the dominant
negative interference response. Dominant negative hypocotyl growth was
measured for ABO and wild-type (RLD) seedlings sown on liquid MS salts
medium (see details in text). Following stratification, seeds were
induced to germinate with a 3-h white-light treatment followed by
21 h in darkness. At that time seeds were transferred to FR light
(8.7 µmol m2 s1), and the medium was
supplemented with a 10× stock of the indicated sugars in MS medium
(2-dG). Hypocotyl measurements were taken after 3 d in FR light.
Error bars indicate ±1 SE of 60 to 120 seedlings from
independent replicate experiments.
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Suc and phyB Effects on phyA Abundance
In attempting to explain the mechanism by which Suc and
ectopically expressed phyB act to inhibit a response normally
attributed to phyA, we used western-blot analysis to examine possible
changes in phyA accumulation and degradation. After 3 d of dark
growth on Suc-containing medium, wild-type and ABO plants were exposed to FR light for varying times or were kept in the dark prior to protein
extraction and SDS-PAGE. After transfer to nitrocellulose, western
blots were probed with monoclonal antibodiesshown previously to
be phyA specific (Hirschfeld et al., 1998)to determine relative initial levels of phyA in Arabidopsis tissues and the comparative rates
of degradation upon exposure to FR light. Figure
6 shows that, within the limits of
quantitation for immunoblotting, the level of PHYA apoprotein in
dark-grown ABO was indistinguishable from that in wild-type seedlings
under our growth conditions.
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| Figure 6.
PhyA accumulation and degradation under FR light.
Seedlings from the transgenic ABO and RLD wild-type (wt) lines were
grown on MS medium supplemented with 2% Suc as described in the text.
Following 2 d of growth in darkness, plates were transferred to FR
light (35 µmol m2 s1) for the indicated
times or were left in darkness for an additional 24 h (Dark).
Seedlings were harvested, and phytochrome-enriched extracts were
subjected to western-blot analysis and probed with a phyA-specific
antibody. Equivalent total proteins (90 µg/gel lane) were loaded onto
gels, and the loading and electroblot transfer was confirmed by Ponceau
S staining. The blot was scanned for the addition of labels and
printing.
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Furthermore, the rate of phyA degradation between 6 and 48 h in FR
light was similar in both lines. By 48 h the amount of phyA (Fig.
6) was essentially undetectable by this method, although there was a
faint signal present in one of the replicate experiments for wild-type
and ABO extracts at 48 h (not shown). We have attributed the
slight shift in the size of the phyA signal to displacement by the
excess phyB present in the ABO samples, rather than to nonspecific
recognition of phyB by the antibodies or degradation of phyA. The 073D
antibodies have been shown to have high specificity for phyA (see
Hirschfeld et al., 1998, and refs. therein), and phyB has a slightly
higher molecular mass than phyA. Furthermore, immunoblots with lower
protein loads do not exhibit this apparent shift in mobility (not
shown).
Dominant Negative Regulation by Photobiologically Inactive
phyB
Among the possible explanations for the dominant negative effects
seen in the ABO line are the following: (a) the excess PHYB apoprotein
competes for limiting chromophore, or (b) phyB-induced signals actively
suppress phyA transduction pathways. To address these possibilities,
the transgenic Cys-357-Ser(No-O) line was analyzed. This line expresses
a PHYB apoprotein in which the Cys chromophore attachment site has been
mutated to a Ser and is known to be photochemically inactive (Wagner et
al., 1996b). Growth of the Cys-357-Ser(No-O) line on Suc-containing
medium was examined at three different fluence rates of FR light (Fig.
7B). At the higher intensities, these
experiments yielded results comparable to those seen in ABO. However,
at the lowest fluence rate tested there was little difference observed
between wild-type No-O and Cys-357-Ser(No-O) seedlings. A photograph of
these seedlings grown at 150 µmol m2
s1 FR light shows that under these
conditions the ABO(RLD) and Cys-357-Ser(No-O) lines appeared identical
to each other but were significantly different from their respective
wild types in hypocotyl length, petiole length, and cotyledon expansion
(Fig. 7A). Quantitation of petiole lengths yielded variable results.
Although the ABO and Cys-357-Ser petioles consistently appeared
slightly longer than those of wild-type plants, technical difficulties
with these measurements resulted in large SDs (not shown).
In the ABO and Cys-357-Ser plants, the cotyledons appeared somewhat
smaller than those of the wild type, similar to previous observations
of phyA (Parks and Quail, 1993). Because of the strong curling observed in cotyledons of FR-light-grown seedlings, quantitation of this phenotype proved unreliable. Root lengths did not vary significantly as
a function of phyB overexpression compared with wild type grown under
comparable conditions (data not shown).
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| Figure 7.
Overexpressed phyB with and without the
chromophore-binding site yield similar dominant negative phenotypes.
Seedlings of parental wild types (RLD and No-O) and transgenics ABO or
phyB with the chromophore-binding site Cys-357 mutated to Ser (C357-S)
were grown for 3 d on MS medium supplemented with 2% Suc at 150 µmol m2 s1 FR light (A) or at the
indicated fluence rates for quantitation of elongation (B). Cys-357-Ser
is in the No-O background, and ABO was introgressed into the RLD
ecotype. Quantitative measurements were made on replicate experiments
consisting of 30 to 50 seedlings per treatment per experiment. Error
bars represent ±1 SE.
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DISCUSSION |
Suc Is Necessary for the Dominant Negative Phenotype of Transgenic
phyB
Studies of mutant Arabidopsis plants deficient in one or more
phytochromes indicate that the strong FR-light suppression of hypocotyl
growth is almost exclusively mediated through the phyA protein (Dehesh
et al., 1993; Nagatani et al., 1993; Parks and Quail, 1993; Reed et
al., 1994). Previously, hypocotyl elongation of phyB-overexpressing
seedlings in FR light has been documented as either identical to that
of wild type (McCormac et al., 1993; Quail et al., 1996) or eliciting a
dominant negative effect on the normal suppression of growth (Wagner et
al., 1996a, 1996b). Among the most probable differences that could
explain this apparent discrepancy was the medium on which the seedlings
were grown. In the former, seedlings were plated on agar containing MS
salts with no added carbohydrates, whereas in the latter, growth medium containing Suc (Valvekens et al., 1988) was used.
Research demonstrating that carbohydrates alter hypocotyl elongation
(Whitelam and Harberd, 1994) and other phytochrome-mediated responses
(Tsukaya et al., 1991; Barnes et al., 1996; Dijkwel et al., 1997) also
indicate a strong interaction between light- and sugar-signaling
pathways. Therefore, we tested the effects of sugar on the
phyB-mediated inhibition of the phyA growth response. As demonstrated
in Figure 1, Suc is a necessary component for obtaining the dominant
negative suppression of phyA-dependent growth inhibition by
constitutively expressed transgenic phyB. Suc was not sufficient to
overcome growth suppression by FR light in the absence of ectopically
expressed phyB, nor was the excess phyB sufficient to block growth
inhibition in the absence of an appropriate sugar. This result implies
interplay, whether direct or indirect, of usable sugars with phyA/phyB
regulatory pathway interactions.
These interference phenomena can be seen throughout the early growth of
the seedlings. At 2, 3, and 4 d after transfer to FR light (Fig.
2), the growth rate appears linear for the wild types and the ABO line.
In contrast, other responses dependent on sugars and phyA show a more
complex relationship. For example, anthocyanin accumulation varies
throughout early development and is dependent on the fluence rate, on
the quality of light, and on the concentration of exogenous sugars
(Mancinelli, 1983; Batschauer et al., 1991; T.W. Short, unpublished
observations). However, FR-light-dependent anthocyanin accumulation is
also decreased in ABO compared with the wild type (T.W. Short,
unpublished observations).
Prior work by Wagner and coworkers (1996b) demonstrated that the
dominant negative phenotype correlates directly with the level of phyB
accumulation in various transgenic lines. This response is fluence rate
dependent (Fig. 3), suggesting that Suc probably acts on the
light-signaling pathway(s) rather than acting solely on the elongation
response. This interpretation is consistent with data from other groups
studying Suc/phytochrome interactions, such as the ability of Suc to
overcome the FR-light-mediated block of greening and expression of the
plastocyanin gene (Dijkwel et al., 1997).
Suc concentration, phyB content, and fluence rate are important for
obtaining a maximal dominant negative response. The growth rate
increases steadily with Suc concentration in the ABO seedlings and, to
a lesser degree, in the wild type. Again, this dosage-dependent result
is more consistent with signal pathway interactions than mere additive
effects on the final response. At higher concentrations of Suc, growth
may be inhibited via a different mechanism. Cell toxicity may be
responsible for the very low germination rates and poor growth at the
highest concentrations tested.
Although the window of fluence rates and the degree of elongation
response we observed differs slightly from those that Dijkwel and
colleagues (1997) reported for wild-type plants harboring a transgenic
plastocyanin-luciferase construct, these small quantitative discrepancies may be explained by differences in: (a) the transgene construct, which may bind to one or more limiting light-regulatory factors; (b) the ecotype background (C24) used in their studies; (c)
the growth medium and Suc concentration used (germination medium
[Valvekens et al., 1988] with or without 3% Suc); and/or (d) the
light-source characteristics and sample size.
Other Metabolizable Sugars Substitute for Suc
Recently, efforts at isolating sugar sensory receptors and
potential signaling pathway genes have been very fruitful. Among the
most promising findings has been the discovery that Arabidopsis hexokinases fulfill metabolic and sensory functions within the plant
(Jang et al., 1997). These hexokinases generally have a broad substrate
specificity, and their regulatory functions are activated by sugars and
sugar analogs that can be phosphorylated (see Koch, 1996).
Monosaccharides such as Man and the Glc analog 2-dG can be
phosphorylated by hexokinase and initiate sensory pathways but are not
metabolized efficiently. This property makes them useful for separating
specific hexokinase-mediated signaling from general carbohydrate status
effects. Although numerous metabolizable sugars, including Glc, Fru,
maltose, and raffinose, were nearly as effective as Suc in promoting
the phyB-dominant negative effect, neither Man nor 2-dG elicited the
response (Fig. 5 and T.W. Short, unpublished observations).
Therefore, it is unlikely that phyA and phyB interactions are modulated
by the hexokinase-signaling pathway. Because comparable concentrations
of mannitol and sorbitol, which are not easily transported into the
plant and are not phosphorylated by hexokinase, were also insufficient
to activate the ABO response, it is unlikely to be a reflection of
general osmotic stress. This conclusion is also consistent with the
apparent specificity of the dominant negative effect. Therefore, the
most likely mechanism of action in this response is either that
phytochrome interactions respond to the general carbohydrate or energy
availability within the plant, or that one or more additional sensory
receptors detect and respond to sugars and have specificities distinct
from those of hexokinase. Although the current experiments cannot
distinguish between these possibilities, evidence from the
sun mutants (Dijkwel et al., 1997) and transgenic sense and
antisense expressors of hexokinases (Jang et al., 1997) indicate that
more than one pathway exists for sugar signaling at the receptor and
downstream transduction steps.
Mechanisms of phyB-Dominant Negative Inhibition of phyA
Signaling
There are many possible explanations for the dominant negative
regulation of phyA pathways by phyB. First, phyB may down-regulate expression or otherwise reduce accumulation of phyA in plant cells. Although phyA has been shown to be present at normal concentrations in
7-d-old etiolated tissue of ABO (Wagner et al., 1996b), its dark
accumulation level and its FR-light-mediated degradation kinetics have
not, to our knowledge, been reported previously for younger tissue. In
the present study we showed that, within the limits of quantitation by
western blotting, the rate of phyA degradation in ABO was
indistinguishable from that of wild-type plants under conditions
similar to those in which the dominant negative response was apparent
(Fig. 6). These results, supported by previous studies showing
comparable phyA levels and photoreversibility under somewhat different
growth conditions, indicate that phyB probably does not repress phyA
production or abundance but, rather, acts by an alternative
mechanism.
Second, the findings shown in Figure 6 imply that excess phyB does not
simply sequester limiting amounts of chromophore. Because degradation
of phyA is dependent on photoconversion to the Pfr form of the molecule
(Vierstra and Quail, 1986; Clough and Vierstra, 1997), comparable
kinetics of phyA turnover between ABO and wild-type seedlings suggest
that the same proportion of PHYA apoprotein molecules binds chromophore
in both lines (Xu et al., 1995). Also consistent with this result, a
mutant ABO with very high levels of photoconvertible phyB but lacking
dominant negative interference of phyA was described by Wagner and
Quail (1995), which shows that binding of chromophore is unlikely to be
the mechanism of interference. Expressed deletion constructs lacking
portions of either the COOH-terminal domain (Boylan et al., 1994;
Cherry and Vierstra, 1994; Wagner et al., 1996b) or the
NH2-terminal domain (including some lacking the
entire chromophore-binding region; Wagner et al., 1996b) have
been found to exhibit a similar dominant negative interference of
phyA. Therefore, either a short region common to
NH2-terminal and COOH-terminal deletion
constructs is responsible for the dominant negative response, or there
are active regions at each end of the molecule, either of which can
give the dominant negative phenotype independently.
Wagner and colleagues (1996b) did not find that the Cys-356-Ser
transgenics caused a dominant negative response. However, closer
examination of the response at different light intensities indicates
that the Cys-356-Ser line does exhibit the phenotype (Fig. 7). At lower
fluence rates, similar to those used in the previous study (6 µmol
m2 s1; Wagner et al.,
1996b), there was essentially no dominant negative effect, but at
higher fluence rates the Cys-356-Ser effect equaled that of ABO.
Together, these data provide evidence that sequestration of chromophore
by ectopic phyB expression is not the primary cause of the ABO FR-light
phenotype.
The above results also suggest a third possibility for explaining the
dominant negative interaction: increased phyB (by virtue of the 18- to
30-fold increase in ABO lines; Wagner et al., 1991) may allow FR-light
activation of phyB pathways that actively inhibit phyA signaling.
Because the photochemically inactive phyB constructas well as
numerous deletion constructsis able to elicit the response, a
photoconvertible product is apparently not requisite, so the effect is
likely passive rather than active in nature.
There are at least two remaining possibilities that represent the most
likely candidates for the observed dominant negative interference
phenotype. Overexpressed phyB may form nonproductive heterodimers with
native phyA protein, effectively reducing the pool of active phyA, or
excess phyB may compete for a common interaction partner, preventing
active phyA homodimers from initiating signaling events. Although the
data presented in this paper cannot differentiate between these
possibilities, there is considerable evidence supporting the second
mechanism over the first. Native gel electrophoresis indicates that
several of the dominant negative constructs are monomeric (Wagner et
al., 1996b). Mutant analysis (Quail et al., 1995; Xu et al., 1995;
Wagner et al., 1996b) and domain-swapping experiments (Wagner et al.,
1996a) have suggested a common signaling mechanism for at least a
subset of phyA and phyB responses. Furthermore, recent results from two
hybrid screens have yielded a gene designated PIF3, whose
product binds to COOH-terminal domains of phyA and phyB (Quail, 1998).
If the latter mechanism of dominant negative interference is accurate,
it is not clear how metabolically active sugars are involved in this
interaction. Among the simplest of the possibilities is that Suc status
affects the relative preference of the phyA/phyB-signaling partner for
one phytochrome versus another by modifying either phytochrome or the
reaction partner. Alternatively, carbohydrates could down-regulate the
overall abundance or binding efficiency of this putative reaction
partner, such that the chance of a productive phyA interaction with the
signaling pathway eventually falls below a threshold for activity. The
coregulation of hypocotyl growth by phytochrome and sugars may be
particularly important for modulating relative growth responsiveness to
phyA and phyB in the context of available storage carbohydrates in the
seed and during the initiation of photosynthesis.
In the presence of sufficient metabolizable sugars at low ratios of red
to FR light, there may be an advantage to the plant in overriding the
normal inhibition of growth (via phyA) and switching rapidly to the
shade-avoidance response mediated through phyB to outcompete
neighboring seedlings. Under lower carbohydrate conditions, the
seedling requires use of existing sugars to produce maximal
photosynthetically active surface area and therefore could be at a
disadvantage if energy were expended in increased elongation.
| |
FOOTNOTES |
1
This work was supported in part by the National
Science Foundation (grant nos. IBN 9421770 and IBN 9734527) and by
grants from The City University of New York Professional Staff
Congress-City University of New York Research Award Program.
*
E-mail timothy_short{at}qc.edu; fax 1-718-997-3445.
Received August 24, 1998;
accepted January 4, 1999.
| |
ABBREVIATIONS |
Abbreviations:
2-dG, 2-deoxyglucose.
ABO, Arabidopsis
phyB-overexpressing.
FR, far-red.
LED, light-emitting diode.
MS, Murashige and Skoog.
phyX, phytochrome X, where X is any letter.
| |
ACKNOWLEDGMENTS |
The author wishes to thank Michael Lin, Peter Altman, and Ana
Maria Estela for their expert technical assistance in these experiments. Special thanks to Dr. Peter Quail for the generous gift of
antibodies and to Drs. Doris Wagner and Peter Quail for transgenic
seeds.
| |
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Abstract
Introduction